Compositions and Methods for Increasing Bone Mineralization

ABSTRACT

A novel class or family of TGF-β binding proteins is disclosed. Also disclosed are assays for selecting molecules for increasing bone mineralization and methods for utilizing such molecules. In particular, compositions and methods relating to antibodies that specifically bind to TGF-beta binding proteins are provided. These methods and compositions relate to altering bone mineral density by interfering with the interaction between a TGF-beta binding protein sclerostin and a TGF-beta superfamily member, particularly a bone morphogenic protein. Increasing bone mineral density has uses in diseases and conditions in which low bone mineral density typifies the condition, such as osteopenia, osteoporosis, and bone fractures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/095,248 (filed Mar. 7, 2002), which is a continuation of U.S.application Ser. No. 09/449,218 (filed Nov. 24, 1999), now issued asU.S. Pat. No. 6,395,511, which claims priority from U.S. ProvisionalApplication No. 60/110,283 filed Nov. 27, 1998. The contents of all theabove applications are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates generally to pharmaceutical products andmethods and, more specifically, to methods and compositions suitable forincreasing the mineral content of bone. Such compositions and methodsmay be utilized to treat a wide variety of conditions, including forexample, osteopenia, osteoporosis, fractures and other disorders inwhich low bone mineral density are a hallmark of the disease.

BACKGROUND OF THE INVENTION

Two or three distinct phases of changes to bone mass occur over the lifeof an individual (see Riggs, West J. Med. 154:63-77, 1991). The firstphase occurs in both men and women, and proceeds to attainment of a peakbone mass. This first phase is achieved through linear growth of theendochondral growth plates, and radial growth due to a rate ofperiosteal apposition. The second phase begins around age 30 fortrabecular bone (flat bones such as the vertebrae and pelvis) and aboutage 40 for cortical bone (e.g., long bones found in the limbs) andcontinues to old age. This phase is characterized by slow bone loss, andoccurs in both men and women. In women, a third phase of bone loss alsooccurs, most likely due to postmenopausal estrogen deficiencies. Duringthis phase alone, women may lose an additional 10% of bone mass from thecortical bone and 25% from the trabecular compartment (see Riggs,supra).

Loss of bone mineral content can be caused by a wide variety ofconditions, and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans characterized by markeddecreases in skeletal bone mass and mineral density, structuraldeterioration of bone including degradation of bone microarchitectureand corresponding increases in bone fragility and susceptibility tofracture in afflicted individuals. Osteoporosis in humans is preceded byclinical osteopenia (bone mineral density that is greater than onestandard deviation but less than 2.5 standard deviations below the meanvalue for young adult bone), a condition found in approximately 25million people in the United States. Another 7-8 million patients in theUnited States have been diagnosed with clinical osteoporosis (defined asbone mineral content greater than 2.5 standard deviations below that ofmature young adult bone). Osteoporosis is one of the most expensivediseases for the health care system, costing tens of billions of dollarsannually in the United States. In addition to health care-related costs,long-term residential care and lost working days add to the financialand social costs of this disease. Worldwide approximately 75 millionpeople are at risk for osteoporosis.

The frequency of osteoporosis in the human population increases withage, and among Caucasians is predominant in women (who comprise 80% ofthe osteoporosis patient pool in the United States). The increasedfragility and susceptibility to fracture of skeletal bone in the aged isaggravated by the greater risk of accidental falls in this population.More than 1.5 million osteoporosis-related bone fractures are reportedin the United States each year. Fractured hips, wrists, and vertebraeare among the most common injuries associated with osteoporosis. Hipfractures in particular are extremely uncomfortable and expensive forthe patient, and for women correlate with high rates of mortality andmorbidity.

Although osteoporosis has been defined as an increase in the risk offracture due to decreased bone mass, none of the presently availabletreatments for skeletal disorders can substantially increase the bonedensity of adults. There is a strong perception among all physiciansthat drugs are needed which could increase bone density in adults,particularly in the bones of the wrist, spinal column and hip that areat risk in osteopenia and osteoporosis.

Current strategies for the prevention of osteoporosis may offer somebenefit to individuals but cannot ensure resolution of the disease.These strategies include moderating physical activity (particularly inweight-bearing activities) with the onset of advanced age, includingadequate calcium in the diet, and avoiding consumption of productscontaining alcohol or tobacco. For patients presenting with clinicalosteopenia or osteoporosis, all current therapeutic drugs and strategiesare directed to reducing further loss of bone mass by inhibiting theprocess of bone absorption, a natural component of the bone remodelingprocess that occurs constitutively.

For example, estrogen is now being prescribed to retard bone loss. Thereis, however, some controversy over whether there is any long termbenefit to patients and whether there is any effect at all on patientsover 75 years old. Moreover, use of estrogen is believed to increase therisk of breast and endometrial cancer.

High doses of dietary calcium, with or without vitamin D has also beensuggested for postmenopausal women. However, high doses of calcium canoften have unpleasant gastrointestinal side effects, and serum andurinary calcium levels must be continuously monitored (see Khosla andRigss, Mayo Clin. Proc. 70:978-982, 1995).

Other therapeutics which have been suggested include calcitonin,bisphosphonates, anabolic steroids and sodium fluoride. Suchtherapeutics however, have undesirable side effects (e.g., calcitoninand steroids may cause nausea and provoke an immune reaction,bisphosphonates and sodium fluoride may inhibit repair of fractures,even though bone density increases modestly) that may prevent theirusage (see Khosla and Rigss, supra).

No currently practiced therapeutic strategy involves a drug thatstimulates or enhances the growth of new bone mass. The presentinvention provides compositions and methods which can be utilized toincrease bone mineralization, and thus may be utilized to treat a widevariety of conditions where it is desired to increase bone mass.Further, the present invention provides other, related advantages.

SUMMARY OF THE INVENTION

As noted above, the present invention provides a novel class or familyof TGF-beta binding-proteins, as well as assays for selecting compoundswhich increase bone mineral content and bone mineral density, compoundswhich increase bone mineral content and bone mineral density and methodsfor utilizing such compounds in the treatment or prevention of a widevariety of conditions.

Within one aspect of the present invention, isolated nucleic acidmolecules are provided, wherein said nucleic acid molecules are selectedfrom the group consisting of: (a) an isolated nucleic acid moleculecomprising sequence ID Nos. 1, 5, 7, 9, 11, 13, or, 15, or complementarysequence thereof; (b) an isolated nucleic acid molecule thatspecifically hybridizes to the nucleic acid molecule of (a) underconditions of high stringency; and (c) an isolated nucleic acid thatencodes a TGF-beta binding-protein according to (a) or (b). Withinrelated aspects of the present invention, isolated nucleic acidmolecules are provided based upon hybridization to only a portion of oneof the above-identified sequences (e.g., for (a) hybridization may be toa probe of at least 20, 25, 50, or 100 nucleotides selected fromnucleotides 156 to 539 or 555 to 687 of Sequence ID No. 1). As should bereadily evident, the necessary stringency to be utilized forhybridization may vary based upon the size of the probe. For example,for a 25-mer probe high stringency conditions could include: 60 mM TrispH 8.0, 2 mM EDTA, 5×Denhardt's, 6×SSC, 0.1% (w/v) N-laurylsarcosine,0.5% (w/v) NP-40 (nonidet P-40) overnight at 45 degrees C., followed bytwo washes with 0.2×SSC/0.1% SDS at 45-50 degrees. For a 100-mer probeunder low stringency conditions, suitable conditions might include thefollowing: 5×SSPE, 5×Denhardt's, and 0.5% SDS overnight at 42-50degrees, followed by two washes with 2×SSPE (or 2×SSC)/0.1% SDS at 42-50degrees.

Within related aspects of the present invention, isolated nucleic acidmolecules are provided which have homology to Sequence ID Nos. 1, 5, 7,9, 11, 13, or 15, at a 50%, 60%, 75%, 80%, 90%, 95%, or 98% level ofhomology utilizing a Wilbur-Lipman algorithm. Representative examples ofsuch isolated molecules include, for example, nucleic acid moleculeswhich encode a protein comprising Sequence ID NOs. 2, 6, 10, 12, 14, or16, or have homology to these sequences at a level of 50%, 60%, 75%,80%, 90%, 95%, or 98% level of homology utilizing a Lipman-Pearsonalgorithm.

Isolated nucleic acid molecules are typically less than 100 kb in size,and, within certain embodiments, less than 50 kb, 25 kb, 10 kb, or even5 kb in size. Further, isolated nucleic acid molecules, within otherembodiments, do not exist in a “library” of other unrelated nucleic acidmolecules (e.g., a subclone BAC such as described in GenBank AccessionNo. AC003098 and EMB No. AQ171546). However, isolated nucleic acidmolecules can be found in libraries of related molecules (e.g., forshuffling, such as is described in U.S. Pat. Nos. 5,837,458; 5,830,721;and 5,811,238). Finally, isolated nucleic acid molecules as describedherein do not include nucleic acid molecules which encode Dan, Cerberus,Gremlin, or SCGF (U.S. Pat. No. 5,780,263).

Also provided by the present invention are cloning vectors which containthe above-noted nucleic acid molecules, and expression vectors whichcomprise a promoter (e.g., a regulatory sequence) operably linked to oneof the above-noted nucleic acid to molecules. Representative examples ofsuitable promoters include tissue-specific promoters, and viral-basedpromoters (e.g., CMV-based promoters such as CMV I-E, SV40 earlypromoter, and MuLV LTR). Expression vectors may also be based upon, orderived from viruses (e.g., a “viral vector”). Representative examplesof viral vectors include herpes simplex viral vectors, adenoviralvectors, adenovirus-associated viral vectors and retroviral vectors.Also provided are host cells containing or comprising any of above-notedvectors (including for example, host cells of human, monkey, dog, rat,or mouse origin).

Within other aspects of the present invention, methods of producingTGF-beta binding-proteins are provided, comprising the step of culturingthe aforementioned host cell containing vector under conditions and fora time sufficient to produce the TGF-beta binding protein. Withinfurther embodiments, the protein produced by this method may be furtherpurified (e.g., by column chromatography, affinity purification, and thelike). Hence, isolated proteins which are encoded by the above-notednucleic acid molecules (e.g., Sequence ID NOs. 2, 4, 6, 8, 10, 12, 14,or 16) may be readily produced given the disclosure of the subjectapplication.

It should also be noted that the aforementioned proteins, or fragmentsthereof, may be produced as fusion proteins. For example, within oneaspect fusion proteins are provided comprising a first polypeptidesegment comprising a TGF-beta binding-protein encoded by a nucleic acidmolecule as described above, or a portion thereof of at least 10, 20,30, 50, or 100 amino acids in length, and a second polypeptide segmentcomprising a non-TGF-beta binding-protein. Within certain embodiments,the second polypeptide may be a tag suitable for purification orrecognition (e.g., a polypeptide comprising multiple anionic amino acidresidues—see U.S. Pat. No. 4,851,341), a marker (e.g., green fluorescentprotein, or alkaline phosphatase), or a toxic molecule (e.g., ricin).

Within another aspect of the present invention, antibodies are providedwhich are capable of specifically binding the above-described class ofTGF-beta binding proteins (e.g., human BEER). Within variousembodiments, the antibody may be a polyclonal antibody, or a monoclonalantibody (e.g., of human or murine origin). Within further embodiments,the antibody is a fragment of an antibody which retains the bindingcharacteristics of a whole antibody (e.g., an F(ab′)₂, F(ab)₂, Fab′,Fab, or Fv fragment, or even a CDR). Also provided are hybridomas andother cells which are capable of producing or expressing theaforementioned antibodies.

Within related aspects of the invention, methods are provided detectinga TGF-beta binding protein, comprising the steps of incubating anantibody as described above under conditions and for a time sufficientto permit said antibody to bind to a TGF-beta binding protein, anddetecting the binding. Within various embodiments the antibody may bebound to a solid support to facilitate washing or separation, and/orlabeled. (e.g., with a marker selected from the group consisting ofenzymes, fluorescent proteins, and radioisotopes).

Within other aspects of the present invention, isolated oligonucleotidesare provided which hybridize to a nucleic acid molecule according toSequence ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, or 18 or the complementthereto, under conditions of high stringency. Within furtherembodiments, the oligonucleotide may be found in the sequence whichencodes Sequence ID Nos. 2, 4, 6, 8, 10, 12, 14, or 16. Within certainembodiments, the oligonucleotide is at least 15, 20, 30, 50, or 100nucleotides in length. Within further embodiments, the oligonucleotideis labeled with another molecule (e.g., an enzyme, fluorescent molecule,or radioisotope). Also provided are primers which are capable ofspecifically amplifying all or a portion of the above-mentioned nucleicacid molecules which encode TGF-beta binding-proteins. As utilizedherein, the term “specifically amplifying” should be understood to referto primers which amplify the aforementioned TGF-beta binding-proteins,and not other TGF-beta binding proteins such as Dan, Cerberus, Gremlin,or SCGF (U.S. Pat. No. 5,780,263).

Within related aspects of the present invention, methods are providedfor detecting a nucleic acid molecule which encodes a TGF-beta bindingprotein, comprising the steps of incubating an oligonucleotide asdescribed above under conditions of high stringency, and detectinghybridization of said oligonucleotide. Within certain embodiments, theoligonucleotide may be labeled and/or bound to a solid support.

Within other aspects of the present invention, ribozymes are providedwhich are capable of cleaving RNA which encodes one of theabove-mentioned TGF-beta binding-proteins (e.g., Sequence ID NOs. 2, 6,8, 10, 12, 14, or 16). Such ribozymes may to be composed of DNA, RNA(including 2′-O-methyl ribonucleic acids), nucleic acid analogs (e.g.,nucleic acids having phosphorothioate linkages) or mixtures thereof.Also provided are nucleic acid molecules (e.g., DNA or cDNA) whichencode these ribozymes, and vectors which are capable of expressing orproducing the ribozymes. Representative examples of vectors includeplasmids, retrotransposons, cosmids, and viral-based vectors (e.g.,viral vectors generated at least in part from a retrovirus, adenovirus,or, adeno-associated virus). Also provided are host cells (e.g., human,dog, rat, or mouse cells) which contain these vectors. In certainembodiments, the host cell may be stably transformed with the vector.

Within further aspects of the invention, methods are provided forproducing ribozymes either synthetically, or by in vitro or in vivotranscription. Within further embodiments, the ribozymes so produced maybe further purified and/or formulated into pharmaceutical compositions(e.g., the ribozyme or nucleic acid molecule encoding the ribozyme alongwith a pharmaceutically acceptable carrier or diluent). Similarly, theantisense oligonucleotides and antibodies or other selected moleculesdescribed herein may be formulated into pharmaceutical compositions.

Within other aspects of the present invention, antisenseoligonucleotides are provided comprising a nucleic acid molecule whichhybridizes to a nucleic acid molecule according to Sequence ID NOs. 1,3, 5, 7, 9, 11, 13, or 15, or the complement thereto, and wherein saidoligonucleotide inhibits the expression of TGF-beta binding protein asdescribed herein (e.g., human BEER). Within various embodiments, theoligonucleotide is 15, 20, 25, 30, 35, 40, or 50 nucleotides in length.Preferably, the oligonucleotide is less than 100, 75, or 60 nucleotidesin length. As should be readily evident, the oligonucleotide may becomprised of one or more nucleic acid analogs, ribonucleic acids, ordeoxyribonucleic acids. Further, the oligonucleotide may be modified byone or more linkages, including for example, covalent linkage such as aphosphorothioate linkage, a phosphotriester linkage, a methylphosphonate linkage, a methylene(methylimino) linkage, a morpholinolinkage, an amide linkage, a polyamide linkage, a short chain alkylintersugar linkage, a cycloalkyl intersugar linkage, a short chainheteroatomic intersugar linkage and a heterocyclic intersugar linkage.One representative example of a chimeric oligonucleotide is provided inU.S. Pat. No. 5,989,912.

Within yet another aspect of the present invention, methods are providedfor increasing bone mineralization, comprising introducing into awarm-blooded animal an effective amount of the ribozyme as describedabove. Within related aspects, such methods comprise the step ofintroducing into a patient an effective amount of the nucleic acidmolecule or vector as described herein which is capable of producing thedesired ribozyme, under conditions favoring transcription of the nucleicacid molecule to produce the ribozyme.

Within other aspects of the invention transgenic, non-human animals areprovided. Within one embodiment a transgenic animal is provided whosegerm cells and somatic cells contain a nucleic acid molecule encoding aTGF-beta binding-protein as described above which is operably linked toa promoter effective for the expression of the gene, the gene beingintroduced into the animal, or an ancestor of the animal, at anembryonic stage, with the proviso that said animal is not a human.Within other embodiments, transgenic knockout animals are provided,comprising an animal whose germ cells and somatic cells comprise adisruption of at least one allele of an endogenous nucleic acid moleculewhich hybridizes to a nucleic acid molecule which encodes a TGF-bindingprotein as described herein, wherein the disruption preventstranscription of messenger RNA from said allele as compared to an animalwithout the disruption, with the proviso that the animal is not a human.Within various embodiments, the disruption is a nucleic acid deletion,substitution, or, insertion. Within other embodiments the transgenicanimal is a mouse, rat, sheep, pig, or dog.

Within further aspects of the invention, kits are provided for thedetection of TGF-beta binding-protein gene expression, comprising acontainer that comprises a nucleic acid molecule, wherein the nucleicacid molecule is selected from the group consisting of (a) a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 100, or 101; (b) a nucleic acid molecule comprisingthe complement of the nucleotide sequence of (a); (c) a nucleic acidmolecule that is a fragment of (a) or (b) of at least 15, 20 30, 50, 75,or, 100 nucleotides in length. Also provided are kits for the detectionof a TGF-beta binding-protein which comprise a container that compriseone of the TGF-beta binding protein antibodies described herein.

For example, within one aspect of the present invention methods areprovided for determining whether a selected molecule is capable ofincreasing bone mineral content, comprising the steps of (a) mixing oneor more candidate molecules with TGF-beta-binding-protein encoded by thenucleic acid molecule according to claim 1 and a selected member of theTGF-beta family of proteins (e.g., BMP 5 or 6), (b) determining whetherthe candidate molecule alters the signaling of the TGF-beta familymember, or alters the binding of the TGF-beta binding-protein to theTGF-beta family member. Within certain embodiments, the molecule altersthe ability of TGF-beta to function as a positive regulator ofmesenchymal cell differentiation. Within this aspect of the presentinvention, the candidate molecule(s) may alter signaling or binding by,for example, either decreasing (e.g., inhibiting), or increasing (e.g.,enhancing) signaling or binding.

Within yet another aspect, methods are provided for determining whethera selected molecule is capable of increasing bone mineral content,comprising the step of determining whether a selected molecule inhibitsthe binding of TGF-beta binding-protein to bone, or an analogue thereof.Representative examples of bone or analogues thereof includehydroxyapatite and primary human bone samples obtained via biopsy.

Within certain embodiments of the above-recited methods, the selectedmolecule is contained within a mixture of molecules and the methods mayfurther comprise the step of isolating one or more molecules which arefunctional within the assay. Within yet other embodiments, TGF-betafamily of proteins is bound to a solid support and the binding ofTGF-beta binding-protein is measured or TGF-beta binding-protein arebound to a solid support and the binding of TGF-beta proteins aremeasured.

Utilizing methods such as those described above, a wide variety ofmolecules may be assayed for their ability to increase bone mineralcontent by inhibiting the binding of the TGF-beta binding-protein to theTGF-beta family of proteins. Representative examples of such moleculesinclude proteins or peptides, organic molecules, and nucleic acidmolecules.

Within other related aspects of the invention, methods are provided forincreasing bone mineral content in a warm-blooded animal, comprising thestep of administering to a warm-blooded animal a therapeuticallyeffective amount of a molecule identified from the assays recitedherein. Within another aspect, methods are provided for increasing bonemineral content in a warm-blooded animal, comprising the step ofadministering to a warm-blooded animal a therapeutically effectiveamount of a molecule which inhibits the binding of the TGF-betabinding-protein to the TGF-beta super-family of proteins, including bonemorphogenic proteins (BMPs). Representative examples of suitablemolecules include antisense molecules, ribozymes, ribozyme genes, andantibodies (e.g., a humanized antibody) which specifically recognize andalter the activity of the TGF-beta binding-protein.

Within another aspect of the present invention, methods are provided forincreasing bone mineral content in a warm-blooded animal, comprising thesteps of (a) introducing into cells which home to the bone a vectorwhich directs the expression of a molecule which inhibits the binding ofthe TGF-beta binding-protein to the TGF-beta family of proteins and bonemorphogenic proteins (BMPs), and (b) administering the vector-containingcells to a warm-blooded animal. As utilized herein, it should beunderstood that cells “home to bone” if they localize within the bonematrix after peripheral administration. Within one embodiment, suchmethods further comprise, prior to the step of introducing, isolatingcells from the marrow of bone which home to the bone. Within a furtherembodiment, the cells which home to bone are selected from the groupconsisting of CD34+ cells and osteoblasts.

Within other aspects of the present invention, molecules are provided(preferably isolated) which inhibit the binding of the TGF-betabinding-protein to the TGF-beta super-family of proteins.

Within further embodiments, the molecules may be provided as acomposition, and can further comprise an inhibitor of bone resorption.Representative examples of such inhibitors include calcitonin, estrogen,a bisphosphonate, a growth factor having anti-resorptive activity andtamoxifen.

Representative examples of molecules which may be utilized in theafore-mentioned therapeutic contexts include, e.g., ribozymes, ribozymegenes, antisense molecules, and/or antibodies (e.g., humanizedantibodies). Such molecules may depending upon their selection, used toalter, antagonize, or agonize the signalling or binding of a TGF-betabinding-protein family member as described herein

Within various embodiments of the invention, the above-describedmolecules and methods of treatment or prevention may be utilized onconditions such as osteoporosis, osteomalacia, periodontal disease,scurvy, Cushing's Disease, bone fracture and conditions due to limbimmobilization and steroid usage.

The present invention also provides antibodies that specifically bind toa TGF-beta binding protein, sclerostin (SOST), and provides immunogenscomprising sclerostin peptides derived from regions of sclerostin thatinteract with a member of the TGF-beta superfamily such as a bonemorphogenic protein. In one embodiment, the invention provides anantibody, or an antigen-binding fragment thereof, that bindsspecifically to a sclerostin polypeptide, said sclerostin polypeptidecomprising an amino acid sequence set forth in SEQ ID NO:2, 6, 8, 14,46, or 65, wherein the antibody competitively inhibits binding of theSOST polypeptide to at least one of (i) a bone morphogenic protein (BMP)Type I Receptor binding site and (ii) a BMP Type II Receptor bindingsite, wherein the BMP Type I Receptor binding site is capable of bindingto a BMP Type I Receptor polypeptide comprising an amino acid sequenceset forth in GenBank Acc. Nos. NM_(—)004329 (SEQ ID NO:102); D89675 (SEQID NO:103); NM_(—)001203 (SEQ ID NO:104); 575359 (SEQ ID NO:105);NM_(—)030849 (SEQ ID NO:106); D38082 (SEQ ID NO:107); NP_(—)001194 (SEQID NO:108); BAA19765 (SEQ ID NO:109); or AAB33865 (SEQ ID NO:110) andwherein the BMP Type II Receptor binding site is capable of binding to aBMP Type II Receptor polypeptide comprising the amino acid sequence setforth in GenBank Acc. NOs. U25110 (SEQ ID NO:111); NM_(—)033346 (SEQ IDNO:112); Z48923 (SEQ ID NO:114); CAA88759 (SEQ ID NO:115); orNM_(—)001204 (SEQ ID NO:113). In another embodiment, the inventionprovides an antibody, or an antigen-binding fragment thereof, that bindsspecifically to a sclerostin polypeptide and that impairs formation of asclerostin homodimer, wherein the sclerostin polypeptide comprises anamino acid sequence set forth in SEQ ID NOs: 2, 6, 8, 14, 46, or 65.

In certain particular embodiments of the invention, the antibody is apolyclonal antibody. In other embodiments, the antibody is a monoclonalantibody, which is a mouse, human, rat, or hamster monoclonal antibody.The invention also provides a hybridoma cell or a host cell that iscapable of producing the monoclonal antibody. In other embodiments ofthe invention, the antibody is a humanized antibody or a chimericantibody. The invention further provides a host cell that produces thehumanized or chimeric antibody. In certain embodiments theantigen-binding fragment of the antibody is a F(ab′)₂, Fab′, Fab, Fd, orFv fragment. The invention also provides an antibody that is a singlechain antibody and provides a host cell that is capable of expressingthe single chain antibody. In another embodiment, the invention providesa composition comprising such antibodies and a physiologicallyacceptable carrier.

In another embodiment, the invention provides an immunogen comprising apeptide comprising at least 21 consecutive amino acids and no more than50 consecutive amino acids of a SOST polypeptide, said SOST polypeptidecomprising an amino acid sequence set forth in SEQ ID NOs: 2, 6, 8, 14,46, or 65, wherein the peptide is capable of eliciting in a non-humananimal an antibody that binds specifically to the SOST polypeptide andthat competitively inhibits binding of the SOST polypeptide to at leastone of (i) a bone morphogenic protein (BMP) Type I Receptor binding siteand (ii) a BMP Type II Receptor binding site, wherein the BMP Type IReceptor binding site is capable of binding to a BMP Type I Receptorpolypeptide comprising an amino acid sequence set forth in GenBank Acc.Nos. NM_(—)004329 (SEQ ID NO:102); D89675 (SEQ ID NO:103); NM_(—)001203(SEQ ID NO:104); 575359 (SEQ ID NO:105); NM_(—)030849 (SEQ ID NO:106);D38082 (SEQ ID NO:107); NP_(—)001194 (SEQ ID NO:108); BAA19765 (SEQ IDNO:109); or AAB33865 (SEQ ID NO:110) and wherein the BMP Type IIReceptor binding site is capable of binding to a BMP Type II Receptorpolypeptide comprising the amino acid sequence set forth in GenBank Acc.NOs. U25110 (SEQ ID NO:111); NM_(—)033346 (SEQ ID NO:112); Z48923 (SEQID NO:114); CAA88759 (SEQ ID NO:115); or NM_(—)001204 (SEQ ID NO:113).The invention also provides an immunogen comprising a peptide thatcomprises at least 21 consecutive amino acids and no more than 50consecutive amino acids of a SOST polypeptide, said SOST polypeptidecomprising an amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 14,46, or 65, wherein the peptide is capable of eliciting in a non-humananimal an antibody that binds specifically to the SOST polypeptide andthat impairs formation of a SOST homodimer.

In certain particular embodiments, the subject invention immunogens areassociated with a carrier molecule. In certain embodiments, the carriermolecule is a carrier polypeptide, and in particular embodiments, thecarrier polypeptide is keyhole limpet hemocyanin.

The invention also provides a method for producing an antibody thatspecifically binds to a SOST polypeptide, comprising immunizing anon-human animal with an immunogen comprising a peptide comprising atleast 21 consecutive amino acids and no more than 50 consecutive aminoacids of a SOST polypeptide, wherein (a) the SOST polypeptide comprisesan amino acid sequence set forth in SEQ ID NO: 2, 6, 8, 14, 46, or 65;(b) the antibody competitively inhibits binding of the SOST polypeptideto at least one of (i) a bone morphogenic protein (BMP) Type I Receptorbinding site and (ii) a BMP Type II Receptor binding site; (c) the BMPType I Receptor binding site is capable of binding to a BMP Type IReceptor polypeptide comprising the amino acid sequence set forth inGenBank Ace. Nos. NM_(—)004329 (SEQ ID NO:102); D89675 (SEQ ID NO:103);NM_(—)001203 (SEQ ID NO:104); S75359 (SEQ ID NO:105); NM_(—)030849 (SEQID NO:106); D38082 (SEQ ID NO:107); NP_(—)001194 (SEQ ID NO:108);BAA19765 (SEQ ID NO:109); or AAB33865 (SEQ ID NO:110); and (d) the BMPType II Receptor binding site is capable of binding to a BMP Type IIReceptor polypeptide comprising the amino acid sequence set forth inGenBank Ace. NOs. U25110 (SEQ ID NO:111); NM_(—)033346 (SEQ ID NO:112);Z48923 (SEQ ID NO:114); CAA88759 (SEQ ID NO:115); or NM_(—)001204 (SEQID NO:113).

In another embodiment, the invention provides a method for producing anantibody that specifically binds to a SOST polypeptide, said SOSTpolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2,6, 8, 14, 46, or 65, comprising immunizing a non-human animal with animmunogen comprising a peptide that comprises at least 21 consecutiveamino acids and no more than 50 consecutive amino acids of a SOSTpolypeptide, said SOST polypeptide comprising an amino acid sequence setforth in SEQ ID NO: 2, 6, 8, 14, 46, or 65, wherein the antibody impairsformation of a SOST homodimer.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, documents including various references set forthherein that describe in more detail certain procedures or compositions(e.g., plasmids, etc.), are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration comparing the amino acid sequence ofHuman Dan; Human Gremlin; Human Cerberus and Human Beer. Arrows indicatethe Cysteine backbone.

FIG. 2 summarizes the results obtained from surveying a variety of humantissues for the expression of a TGF-beta binding-protein gene,specifically, the Human Beer gene. A semi-quantitative ReverseTranscription-Polymerase Chain Reaction (RT-PCR) procedure was used toamplify a portion of the gene from first-strand cDNA synthesized fromtotal RNA (described in more detail in EXAMPLE 2A).

FIGS. 3A-3D summarize the results obtained from RNA in situhybridization of mouse embryo sections, using a cRNA probe that iscomplementary to the mouse Beer transcript (described in more detail inEXAMPLE 2B). Panel 3A is a transverse section of 10.5 dpc embryo. Panel3B is a sagittal section of 12.5 dpc embryo and panels 3C and 3D aresagittal sections of 15.5 dpc embryos.

FIGS. 4A-4C illustrate, by western blot analysis, the specificity ofthree different polyclonal antibodies for their respective antigens(described in more detail in EXAMPLE 4). FIG. 4A shows specificreactivity of an anti-H. Beer antibody for H. Beer antigen, but not H.Dan or H. Gremlin. FIG. 4B shows reactivity of an anti-H. Gremlinantibody for H. Gremlin antigen, but not H. Beer or H. Dan. FIG. 4Cshows reactivity of an anti-H. Dan antibody for H. Dan, but not H. Beeror H. Gremlin.

FIG. 5 illustrates, by western blot analysis, the selectivity of theTGF-beta binding-protein, Beer, for BMP-5 and BMP-6, but not BMP-4(described in more detail in EXAMPLE 5).

FIG. 6 demonstrates that the ionic interaction between the TGF-betabinding-protein, Beer, and BMP-5 has a dissociation constant in the15-30 nM range.

FIG. 7 presents an alignment of the region containing the characteristiccystine-knot of a SOST (sclerostin) polypeptide and its closesthomologues. Three disulphide bonds that form the cystine-knot areillustrated as solid lines. An extra disulphide bond, shown by a dottedline, is unique to this family, which connects two β-hairpin tips in the3D structure. The polypeptides depicted are SOST: sclerostin (SEQ IDNO:126); CGHB: Human Chorionic Gonadotropin β (SEQ ID NO:127); FSHB:follicle-stimulating hormone beta subunit (SEQ ID NO:128); TSHB:thyrotropin beta chain precursor (SEQ ID NO:129); VWF: Von Willebrandfactor (SEQ ID NO:130); MUC2: human mucin 2 precursor (SEQ ID NO:131);CER1: Cerberus 1 (Xenopus laevis homolog) (SEQ ID NO:132); DRM: gremlin(SEQ ID NO:133); DAN: (SEQ ID NO:134); CTGF: connective tissue growthfactor precursor (SEQ ID NO:135); NOV: NovH (nephroblastomaoverexpressed gene protein homolog) (SEQ ID NO:136); CYR6: (SEQ IDNO:137).

FIG. 8 illustrates a 3D model of the core region of SOST (SOST_Core).

FIG. 9 presents a 3D model of the core region of SOST homodimer.

FIGS. 10A and 10B provide an amino acid sequence alignment of Nogginfrom five different animals: human (NOGG_HUMAN (SEQ ID NO:138); chicken(NOGG_CHICK, SEQ ID NO:139); African clawed frog (NOGG_XENLA, SEQ IDNO:140); NOGG_FUGRU, SEQ ID NO:141); and zebrafish (NOGG_ZEBRA, SEQ IDNO:142); and SOST from human (SOST_HUMAN, SEQ ID NO:46), rat (SOST_RAT,SEQ ID NO:65), and mouse (SOST_Mouse, SEQ ID NO:143).

FIG. 11 illustrates the Noggin/BMP-7 complex structure. The BMPhomodimer is shown on the bottom portion of the figure in surface mode.The Noggin homodimer is shown on top of the BMP dimer in cartoon mode.The circles outline the N-terminal binding region, the core region, andthe linker between the N-terminal and core regions.

FIG. 12 depicts a 3D model of the potential BMP-binding fragment locatedat the SOST N-terminal region. A BMP dimer is shown in surface mode, andthe potential BMP-binding fragment is shown in stick mode. Aphenylalanine residue fitting into a hydrophobic pocket on the BMPsurface is noted.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Prior to setting forth the invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms and tolist and to define the abbreviations that will be used hereinafter.

“Molecule” should be understood to include proteins or peptides (e.g.,antibodies, recombinant binding partners, peptides with a desiredbinding affinity), nucleic acids (e.g., DNA, RNA, chimeric nucleic acidmolecules, and nucleic acid analogues such as PNA); and organic orinorganic compounds.

“TGF-beta” should be understood to include any known or novel member ofthe TGF-beta super-family, which also includes bone morphogenic proteins(BMPs).

“TGF-beta receptor” should be understood to refer to the receptorspecific for a particular member of the TGF-beta super-family (includingbone morphogenic proteins (BMPs)).

“TGF-beta binding-protein” should be understood to refer to a proteinwith specific binding affinity for a particular member or subset ofmembers of the TGF-beta super-family (including bone morphogenicproteins (BMPs)). Specific examples of TGF-beta binding-proteins includeproteins encoded by Sequence ID Nos. 1, 5, 7, 9, 11, 13, 15, 100, and101.

Inhibiting the “binding of the TGF-beta binding-protein to the TGF-betafamily of proteins and bone morphogenic proteins (BMPs)” should beunderstood to refer to molecules which allow the activation of TGF-betaor bone morphogenic proteins (BMPs), or allow the binding of TGF-betafamily members including bone morphogenic proteins (BMPs) to theirrespective receptors, by removing or preventing TGF-beta from binding toTGF-binding-protein. Such inhibition may be accomplished, for example,by molecules which inhibit the binding of the TGF-beta binding-proteinto specific members of the TGF-beta super-family.

“Vector” refers to an assembly that is capable of directing theexpression of desired protein. The vector must include transcriptionalpromoter elements that are operably linked to the gene(s) of interest.The vector may be composed of deoxyribonucleic acids (“DNA”),ribonucleic acids (“RNA”), or a combination of the two (e.g., a DNA-RNAchimeric). Optionally, the vector may include a polyadenylationsequence, one or more restriction sites, as well as one or moreselectable markers such as neomycin phosphotransferase or hygromycinphosphotransferase. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements such as an origin ofreplication, additional nucleic acid restriction sites, enhancers,sequences conferring inducibility of transcription, and selectablemarkers, may also be incorporated into the vectors described herein.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a TGF-binding protein that has been separated fromthe genomic DNA of a eukaryotic cell is an isolated DNA molecule.Another example of an isolated nucleic acid molecule is achemically-synthesized nucleic acid molecule that is not integrated inthe genome of an organism. The isolated nucleic acid molecule may begenomic DNA, cDNA, RNA, or composed at least in part of nucleic acidanalogs.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Preferably, such isolated polypeptides are at least about 90% pure, morepreferably at least about 95% pure, and most preferably at least about99% pure. Within certain embodiments, a particular protein preparationcontains an isolated polypeptide if it appears nominally as a singleband on SDS-PAGE gel with Coomassie Blue staining. The term “isolated”when referring to organic molecules (e.g., organic small molecules)means that the compounds are greater than 90% pure utilizing methodswhich are well known in the art (e.g., NMR, melting point).

“Sclerosteosis” is a term that was applied by Hansen (1967) (Hansen, H.G., Sklerosteose. in: Opitz, H.; Schmid, F., Handbuch derKinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351-355) to adisorder similar to van Buchem hyperostosis corticalis generalisata butpossibly differing in radiologic appearance of the bone changes and inthe presence of asymmetric cutaneous syndactyly of the index and middlefingers in many cases. The jaw has an unusually square appearance inthis condition.

“Humanized antibodies” are recombinant proteins in which murine or othernon-human animal complementary determining regions of monoclonalantibodies have been transferred from heavy and light variable chains ofthe murine or other non-human animal immunoglobulin into a humanvariable domain.

As used herein, an “antibody fragment” is a portion of an antibody suchas F(ab′)₂, F(ab)₂, Fab′, Fab, and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-TGF-beta binding-proteinmonoclonal antibody fragment binds to an epitope of TGF-betabinding-protein.

The term antibody fragment or antigen-binding fragment also includes anysynthetic or genetically engineered protein that acts like an antibodyby binding to a specific antigen to form a complex. For example,antibody fragments include isolated fragments consisting of the lightchain variable region, “Fv” fragments consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“sFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion.

A “detectable label” is a molecule or atom that can be conjugated to apolypeptide moiety such as an antibody moiety or a nucleic acid moietyto produce a molecule useful for diagnosis. Examples of detectablelabels include chelators, photoactive agents, radioisotopes, fluorescentagents, paramagnetic ions, enzymes, and other marker moieties.

As used herein, an “immunoconjugate” is a molecule comprising ananti-TGF-beta binding-protein antibody, or an antibody fragment, and adetectable label or an effector molecule. Preferably, an immunoconjugatehas roughly the same, or only slightly reduced, ability to bind TGF-betabinding-protein after conjugation as before conjugation.

Abbreviations: TGF-beta—“Transforming Growth Factor-beta”;TGF-bBP—“Transforming Growth Factor-beta binding-protein” (onerepresentative TGF-bBP is designated “H. Beer”); BMP—“bone morphogenicprotein”; PCR—“polymerase chain reaction”; RT-PCR—PCR process in whichRNA is first transcribed into DNA using reverse transcriptase (RT);cDNA—any DNA made by copying an RNA sequence into DNA form.

As noted above, the present invention provides a novel class of TGF-betabinding-proteins, as well as methods and compositions for increasingbone mineral content in warm-blooded animals. Briefly, the presentinventions are based upon the unexpected discovery that a mutation inthe gene which encodes a novel member of the TGF-beta binding-proteinfamily results in a rare condition (sclerosteosis) characterized by bonemineral contents which are one- to four-fold higher than in normalindividuals. Thus, as discussed in more detail below this discovery hasled to the development of assays which may be utilized to selectmolecules which inhibit the binding of the TGF-beta binding-protein tothe TGF-beta family of proteins and bone morphogenic proteins (BMPs),and methods of utilizing such molecules for increasing the bone mineralcontent of warm-blooded animals (including for example, humans).

Discussion of the Disease Known as Sclerosteosis

Sclerosteosis is a disease related to abnormal bone mineral density inhumans. Sclerosteosis is a term that was applied by Hansen (1967)(Hansen, H. G., Sklerosteose. In: Opitz, H.; Schmid, F., Handbuch derKinderheilkunde. Berlin: Springer (pub.) 6 1967. Pp. 351-355) to adisorder similar to van Buchem hyperostosis corticalis generalisata butpossibly differing in radiologic appearance of the bone changes anddiffering in the presence of asymmetric cutaneous syndactyly of theindex and middle fingers in many cases.

Sclerosteosis is now known to be an autosomal semi-dominant disorderthat is characterized by widely disseminated sclerotic lesions of thebone in the adult. The condition is progressive. Sclerosteosis also hasa developmental aspect that is associated with syndactyly (two or morefingers are fused together). The Sclerosteosis Syndrome is associatedwith large stature and many affected individuals attain a height of sixfeet or more. The bone mineral content of homozygotes can be 1 to 6 foldgreater than observed in normal individuals, and bone mineral densitycan be 1 to 4 fold above normal values (e.g., from unaffected siblings).

The Sclerosteosis Syndrome occurs primarily in Afrikaaners of Dutchdescent in South Africa. Approximately 1/140 individuals in theAfrikaaner population are carriers of the mutated gene (heterozygotes):The mutation shows 100% penetrance. There are anecdotal reports ofincreased of bone mineral density in heterozygotes with no associatedpathologies (syndactyly or skull overgrowth).

No abnormality of the pituitary-hypothalamus axis has been observed inpatients with sclerosteosis. In particular, there appears to be noover-production of growth hormone and cortisone. In addition, sexhormone levels are normal in affected individuals. However, boneturnover markers (osteoblast specific alkaline phosphatase, osteocalcin,type 1 procollagen C′ propeptide (PICP), and total alkaline phosphatase;(see Cornier, C., Curr. Opin. in Rheu. 7:243, 1995) indicate that thereis hyperosteoblastic activity associated with the disease but that thereis normal to slightly decreased osteoclast activity as measured bymarkers of bone resorption (pyridinoline, deoxypyridinoline,N-telopeptide, urinary hydroxyproline, plasma tartrate-resistant acidphosphatases and galactosyl hydroxylysine (see Comier, supra)).

Sclerosteosis is characterized by the continual deposition of bonethroughout the skeleton during the lifetime of the affected individuals.In homozygotes the continual deposition of bone mineral leads to anovergrowth of bone in areas of the skeleton where there is an absence ofmechanoreceptors (skull, jaw, cranium). In homozygotes withSclerosteosis, the overgrowth of the bones of the skull leads to cranialcompression and eventually to death due to excessive hydrostaticpressure on the brain stem. In all other parts of the skeleton there isa generalized and diffuse sclerosis. Cortical areas of the long bonesare greatly thickened resulting in a substantial increase in bonestrength. Trabecular connections are increased in thickness which inturn increases the strength of the trabecular bone. Sclerotic bonesappear unusually opaque to x-rays.

As described in more detail in Example 1, the rare genetic mutation thatis responsible for the Sclerosteosis syndrome has been localized to theregion of human chromosome 17 that encodes a novel member of theTGF-beta binding-protein family (one representative example of which isdesignated “H. Beer”). As described in more detail below, based uponthis discovery, the mechanism of bone mineralization is more fullyunderstood, allowing the development of assays for molecules thatincrease bone mineralization, and use of such molecules to increase bonemineral content, and in the treatment or prevention of a wide number ofdiseases.

TGF-Beta Super-Family

The Transforming Growth Factor-beta (TGF-beta) super-family contains avariety of growth factors that share common sequence elements andstructural motifs (at both the secondary and tertiary levels). Thisprotein family is known to exert a wide spectrum of biological responsesthat affect a large variety of cell types. Many of the TGF-beta familymembers have important functions during the embryonal development inpattern formation and tissue specification; in adults the family membersare involved, e.g., in wound healing and bone repair and boneremodeling, and in the modulation of the immune system. In addition tothe TGF-beta's, the super-family includes the Bone Morphogenic Proteins(BMPs), Activins, Inhibins, Growth and Differentiation Factors (GDFs),and Glial-Derived Neurotrophic Factors (GDNFs). Primary classificationis established through general sequence features that bin a specificprotein into a general sub-family. Additional stratification within thesub-family is possible due to stricter sequence conservation betweenmembers of the smaller group. In certain instances, such as with BMP-5,BMP-6 and BMP-7, the amino acid identity can be as high as 75% amongmembers of the smaller group. This level of identity enables a singlerepresentative sequence to illustrate the key biochemical elements ofthe sub-group that separates it from other members of the larger family.

The crystal structure of TGF-beta2 has been determined. The general foldof the TGF-beta2 monomer contains a stable, compact, cysteine knotlikestructure formed by three disulphide bridges. Dimerization, stabilizedby one disulfide bridge, is antiparallel.

TGF-beta signals by inducing the formation of hetero-oligomericcomplexes of type I and type II receptors. Transduction of TGF-betasignals involves these two distinct type I and type II subfamilies oftransmembrane serine/threonine kinase receptors. At least seven type Ireceptors and five type II receptors have been identified (see Kawabataet al., Cytokine Growth Factor Rev. 9:49-61 (1998); Miyazono et al.,Adv. Immunol. 75:115-57 (2000). TGF-beta family members initiate theircellular action by binding to receptors with intrinsic serine/threoninekinase activity. Each member of the TGF-beta family binds to acharacteristic combination of type I and type II receptors, both ofwhich are needed for signaling. In the current model for TGF-betareceptor activation, a TGF-beta ligand first binds to the type IIreceptor (TbR-II), which occurs in the cell membrane in an oligomericform with activated kinase. Thereafter, the type I receptor (TbR-I),which cannot bind ligand in the absence of TbR-II, is recruited into thecomplex to form a ligand/type II/type I ternary complex. TbR-II thenphosphorylates TbR-I predominantly in a domain rich in glycine andserine residues (GS domain) in the juxtamembrane region, and therebyactivates TbR-I. The activated type I receptor kinase thenphosphorylates particular members of the Smad family of proteins thattranslocate to the nucleus where they modulate transcription of specificgenes.

Bone Morphogenic Proteins (BMPs) are Key Regulatory Proteins inDetermining Bone Mineral Density in Humans

A major advance in the understanding of bone formation was theidentification of the bone morphogenic proteins (BMPs), also known asosteogenic proteins (OPs), which regulate cartilage and bonedifferentiation in vivo. BMPs/OPs induce endochondral bonedifferentiation through a cascade of events that include formation ofcartilage, hypertrophy and calcification of the cartilage, vascularinvasion, differentiation of osteoblasts, and formation of bone. Asdescribed above, the BMPs/OPs (BMP 2-14, and osteogenic protein 1 and-2, OP-1 and OP-2) see, e.g., GenBank P12643 (BMP-2); GenBank P12645(BMP3); GenBank P55107 (BMP-3b, Growth/differentiation factor 10)(GDF-10)); GenBank P12644 (BMP4); GenBank P22003 (BMP5); GenBank P22004(BMP6); GenBank P18075 (BMP7); GenBank P34820 (BMP8); GenBank Q9UK05(BMP9); GenBank O95393 (BM10); GenBank O95390 (BMP11,Growth/differentiation factor 11 precursor (GDF-11)); GenBank O95972(BM15)) are members of the TGF-beta super-family. The strikingevolutionary conservation between members the BMP/OP sub-family suggeststhat they are critical in the normal development and function ofanimals. Moreover, the presence of multiple forms of BMPs/OPs raises animportant question about the biological relevance of this apparentredundancy. In addition to postfetal chondrogenesis and osteogenesis,the BMPs/OPs play multiple roles in skeletogenesis (including thedevelopment of craniofacial and dental tissues) and in embryonicdevelopment and organogenesis of parenchymatous organs, including thekidney. It is now understood that nature relies on common (and few)molecular mechanisms tailored to provide the emergence of specializedtissues and organs. The BMP/OP super-family is an elegant example ofnature parsimony in programming multiple specialized functions deployingmolecular isoforms with minor variation in amino acid motifs withinhighly conserved carboxy-terminal regions.

BMPs are synthesized as large precursor proteins. Upon dimerization, theBMPs are proteolytically cleaved within the cell to yieldcarboxy-terminal mature proteins that are then secreted from the cell.BMPs, like other TGF-beta family members, initiate signal transductionby binding cooperatively to both type I and type II serine/threoninekinase receptors. Type I receptors for which BMPs may act as ligandsinclude BMPR-IA (also known as ALK-3), BMPR-IB (also known as ALK-6),ALK-1, and ALK-2 (also known as ActR-I). Of the type II receptors, BMPsbind to BMP type II receptor (BMPR-II), Activin type II (ActR-II), andActivin type IIB (ActR-IIB). (See Balemans et al., supra, and referencescited therein). Polynucleotide sequences and the encoded amino acidsequence of BMP type I receptor polypeptides are provided in the GenBankdatabase, for example, GenBank NM_(—)004329 (SEQ ID NO:102 encoded bySEQ ID NO:116); D89675 (SEQ ID NO:103 encoded by SEQ ID NO:117);NM_(—)001203 (SEQ ID NO:104 encoded by SEQ ID NO:118); 575359 (SEQ IDNO:105 encoded by SEQ ID NO:119); NM_(—)030849 (SEQ ID NO:106 encoded bySEQ ID NO:120); and D38082 (SEQ ID NO:107 encoded by SEQ ID NO:121).Other polypeptide sequences of type I receptors are provided in theGenBank database, for example, NP_(—)001194 (SEQ ID NO:108); BAA19765(SEQ ID NO:109); and AAB33865 (SEQ ID NO:110). Polynucleotide sequencesand the encoded amino acid sequence of BMP type II receptor polypeptidesare provided in the GenBank database and include, for example, U25110(SEQ ID NO:111 encoded by SEQ ID NO:122); NM_(—)033346 (SEQ ID NO:112encoded by SEQ ID NO:123); NM_(—)001204 (SEQ ID NO:113 encoded by SEQ IDNO:124); and Z48923 (SEQ ID NO:114 encoded by SEQ ID NO:125). Additionalpolypeptide sequences of type II receptors are also provided in theGenBank database, for example, CAA88759 (SEQ ID NO:115).

BMPs, similar to other cystine-knot proteins, form a homodimer structure(Scheufler et al., J. Mol. Biol. 287:103-15 (1999)). According toevolutionary trace analysis performed on the BMP/TGF-β family, the BMPtype I receptor binding site and type II receptor binding site weremapped to the surface of the BMP structure (Innis et al., Protein Eng.13:839-47 (2000)). The location of the type I receptor binding site onBMP was later confirmed by the x-ray structure of BMP-2/BMP Receptor IAcomplex (Nickel et al., J. Joint Surg. Am. 83A(Suppl 1(Pt 1)):S7-S14(2001)). The predicted type II receptor binding site is in goodagreement with the x-ray structure of TGF-β3/TGF-β Type II receptorcomplex (Hart et al., Nat. Struct. Biol. 9:203-208 (2002)), which ishighly similar to the BMP/BMP Receptor IIA system.

BMP Antagonism

The BMP and Activin sub-families are subject to significantpost-translational regulation, such as by TGF-beta binding proteins. Anintricate extracellular control system exists, whereby a high affinityantagonist is synthesized and exported, and subsequently complexesselectively with BMPs or activins to disrupt their biological activity(W. C. Smith (1999) TIG 15(1) 3-6). A number of these naturalantagonists have been identified, and on the basis of sequencedivergence, the antagonists appear to have evolved independently due tothe lack of primary sequence conservation. Earlier studies of theseantagonists highlighted a distinct preference for interacting andneutralizing BMP-2 and BMP-4. In vertebrates, antagonists includenoggin, chordin, chordin-like, follistatin, FSRP, the DAN/Cerberusprotein family, and sclerostin (SOST) (see Balemans et al., supra, andreferences cited therein). The mechanism of antagonism or inhibitionseems to differ for the different antagonists (Iemura et al. (1998)Proc. Natl. Acad. Sci. USA 95 9337-9342).

The type I and type II receptor binding sites on the BMP antagonistnoggin have also been mapped. Noggin binds to BMPs with high affinity(Zimmerman et al., 1996). A study of the noggin/BMP-7 complex structurerevealed the binding interactions between the two proteins (Groppe etal., Nature 420:636-42 (2002)). Superposition of the noggin-BMP-7structure onto a model of the BMP signaling complex showed that nogginbinding effectively masks both pairs of binding epitopes (i.e., BMP TypeI and Type II receptor binding sites) on BMP-7. The cysteine-richscaffold sequence of noggin is preceded by an N-terminal segment ofabout 20 amino acid residues that are referred to as the “clip”(residues 28-48). The type I receptor-binding site is occluded by theN-terminal portion of the clip domain of Noggin, and the type IIreceptor binding site is occluded by the carboxy terminal portion of theclip domain. Two O-strands in the core region near the C-terminus ofnoggin also contact BMP-7 at the type II receptor binding site. Thisbinding mode enables a noggin dimer to efficiently block all thereceptor binding sites (two type I and two type II receptor bindingsites) on a BMP dimer.

Novel TGF-Beta Binding-Proteins

As noted above, the present invention provides a novel class of TGF-betabinding-proteins that possess a nearly identical cysteine (disulfide)scaffold when compared to Human DAN, Human Gremlin, and Human Cerberus,and SCGF (U.S. Pat. No. 5,780,263) but almost no homology at thenucleotide level (for background information, see generally Hsu, D. R.,Economides, A. N., Wang, X., Eimon, P. M., Harland, R. M., “The XenopusDorsalizing Factor Gremlin Identifies a Novel Family of SecretedProteins that Antagonize BMP Activities,” Molecular Cell 1:673-683,1998).

Representative example of the novel class of nucleic acid moleculesencoding TGF-beta binding-proteins are disclosed in SEQ ID NOs: 1, 5, 7,9, 11, 13, 15, 100, and 101. The polynucleotides disclosed herein encodea polypeptide called Beer, which is also referred to herein assclerostin or SOST. Representative members of this class of bindingproteins should also be understood to include variants of the TGF-betabinding-protein (e.g., SEQ ID NOs: 5 and 7). As utilized herein, a“TGF-beta binding-protein variant gene” (e.g., an isolated nucleic acidmolecule that encodes a TGF-beta binding protein variant) refers tonucleic acid molecules that encode a polypeptide having an amino acidsequence that is a modification of SEQ ID Nos: 2, 10, 12, 14, 16, 46, or65. Such variants include naturally-occurring polymorphisms or allelicvariants of TGF-beta binding-protein genes, as well as synthetic genesthat contain conservative amino acid substitutions of these amino acidsequences. A variety of criteria known to those skilled in the artindicate whether amino acids at a particular position in a peptide orpolypeptide are similar. For example, a similar amino acid or aconservative amino acid substitution is one in which an amino acidresidue is replaced with an amino acid residue having a similar sidechain, which include amino acids with basic side chains (e.g., lysine,arginine, histidine); acidic side chains (e.g., aspartic acid, glutamicacid); uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, histidine); nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); beta-branched side chains (e.g.,threonine, valine, isoleucine), and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan). Proline, which is considered moredifficult to classify, shares properties with amino acids that havealiphatic side chains (e.g., Leu, Val, Ile, and Ala). In certaincircumstances, substitution of glutamine for glutamic acid or asparaginefor aspartic acid may be considered a similar substitution in thatglutamine and asparagine are amide derivatives of glutamic acid andaspartic acid, respectively.

Additional variant forms of a TGF-beta binding-protein gene are nucleicacid molecules that contain insertions or deletions of the nucleotidesequences described herein. TGF-beta binding-protein variant genes canbe identified by determining whether the genes hybridize with a nucleicacid molecule having the nucleotide sequence of SEQ ID Nos: 1, 5, 7, 9,11, 13, 15, 100, or 101 under stringent conditions. In addition,TGF-beta binding-protein variant genes should encode a protein having acysteine backbone.

As an alternative, TGF-beta binding-protein variant genes can beidentified by sequence comparison. As used herein, two amino acidsequences have “100% amino acid sequence identity” if the amino acidresidues of the two amino acid sequences are the same when aligned formaximal correspondence. Similarly, two nucleotide sequences have “100%nucleotide sequence identity” if the nucleotide residues of the twonucleotide sequences are the same when aligned for maximalcorrespondence. Sequence comparisons can be performed using standardsoftware programs such as those included in the LASERGENE bioinformaticscomputing suite, which is produced by DNASTAR (Madison, Wis.). Othermethods for comparing two nucleotide or amino acid sequences bydetermining optimal alignment are well-known to those of skill in theart (see, for example, Peruski and Peruski, The Internet and the NewBiology: Tools for Genomic and Molecular Research (ASM Press, Inc.1997), Wu et al. (eds.), “Information Superhighway and ComputerDatabases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)).

A variant TGF-beta binding-protein should have at least a 50% amino acidsequence identity to SEQ ID NOs: 2, 6, 10, 12, 14, 16, 46, or 65 andpreferably, greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity. Alternatively, TGF-beta binding-protein variants can beidentified by having at least a 70% nucleotide sequence identity to SEQID NOs: 1, 5, 9, 11, 13, 15, 100, or 101. Moreover, the presentinvention contemplates TGF-beta binding-protein gene variants havinggreater than 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:1 or SEQID NO:100. Regardless of the particular method used to identify aTGF-beta binding-protein variant gene or variant TGF-betabinding-protein, a variant TGF-beta binding-protein or a polypeptideencoded by a variant TGF-beta binding-protein gene can be functionallycharacterized by, for example, its ability to bind to and/or inhibit thesignaling of a selected member of the TGF-beta family of proteins, or byits ability to bind specifically to an anti-TGF-beta binding-proteinantibody.

The present invention includes functional fragments of TGF-betabinding-protein genes. Within the context of this invention, a“functional fragment” of a TGF-beta binding-protein gene refers to anucleic acid molecule that encodes a portion of a TGF-betabinding-protein polypeptide which either (1) possesses the above-notedfunction activity, or (2) specifically binds with an anti-TGF-betabinding-protein antibody. For example, a functional fragment of aTGF-beta binding-protein gene described herein comprises a portion ofthe nucleotide sequence of SEQ ID Nos: 1, 5, 9, 11, 13, 15, 100, or 101.

2. Isolation of the TGF-Beta Binding-Protein Gene

DNA molecules encoding a TGF-beta binding-protein can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon, for example, SEQ ID NO:1. For example, the first step in thepreparation of a cDNA library is to isolate RNA using methods well-knownto those of skill in the art. In general, RNA isolation techniquesprovide a method for breaking cells, a means of inhibitingRNase-directed degradation of RNA, and a method of separating RNA fromDNA, protein, and polysaccharide contaminants. For example, total RNAcan be isolated by freezing tissue in liquid nitrogen, grinding thefrozen tissue with a mortar and pestle to lyse the cells, extracting theground tissue with a solution of phenol/chloroform to remove proteins,and separating RNA from the remaining impurities by selectiveprecipitation with lithium chloride (see, for example, Ausubel et al.(eds.), Short Protocols in Molecular Biology, 3rd Edition, pages 4-1 to4-6 (John Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al., Methods inGene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [“Wu (1997)”]).Alternatively, total RNA can be isolated by extracting ground tissuewith guanidinium isothiocyanate, extracting with organic solvents, andseparating RNA from contaminants using differential centrifugation (see,for example, Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages33-41).

In order to construct a cDNA library, poly(A)⁺RNA is preferably isolatedfrom a total RNA preparation. Poly(A)⁺ RNA can be isolated from totalRNA by using the standard technique of oligo(dT)-cellulosechromatography (see, for example, Ausubel (1995) at pages 4-11 to 4-12).Double-stranded cDNA molecules may be synthesized from poly(A)⁺RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules (for example, LifeTechnologies, Inc. (Gaithersburg, Md.); CLONTECH Laboratories, Inc.(Palo Alto, Calif.); Promega Corporation (Madison, Wis.); and StratageneCloning Systems (La Jolla, Calif.)).

The basic approach for obtaining TGF-beta binding-protein cDNA clonescan be modified by constructing a subtracted cDNA library that isenriched in TGF-binding-protein-specific cDNA molecules. Techniques forconstructing subtracted libraries are well-known to those of skill inthe art (see, for example, Sargent, “Isolation of DifferentiallyExpressed Genes,” in Meth. Enzymol. 152:423, 1987; and Wu et al. (eds.),“Construction and Screening of Subtracted and Complete Expression cDNALibraries,” in Methods in Gene Biotechnology, pages 29-65 (CRC Press,Inc. 1997)).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector (see, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52). Alternatively, double-strandedcDNA molecules can be inserted into a plasmid vector, such as apBluescript vector (Stratagene Cloning Systems; La Jolla, Calif.), aLambdaGEM-4 (Promega Corp.; Madison, Wis.) or other commerciallyavailable vectors. Suitable cloning vectors also can be obtained fromthe American Type Culture Collection (Rockville, Md.).

In order to amplify the cloned cDNA molecules, the cDNA library isinserted into a prokaryotic host, using standard techniques. Forexample, a cDNA library can be introduced into competent E. coli DH5cells, which can be obtained from Life Technologies, Inc. (Gaithersburg,Md.).

A human genomic DNA library can be prepared by means well-known in theart (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Nucleic acid molecules that encode a TGF-beta binding-protein can alsobe obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences of the human TGF-beta binding-protein gene, asdescribed herein. General methods for screening libraries with PCR areprovided by, for example, Yu et al., “Use of the Polymerase ChainReaction to Screen Phage Libraries,” in Methods in Molecular Biology,Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.),pages 211-215 (Humana Press, Inc. 1993). Moreover, techniques for usingPCR to isolate related genes are described by, for example, Preston,“Use of Degenerate Oligonucleotide Primers and the Polymerase ChainReaction to Clone Gene Family Members,” in Methods in Molecular Biology,Vol. 15: PCR Protocols: Current Methods and Applications, White (ed),pages 317-337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Rockville, Md.). A library containing cDNA orgenomic clones can be screened with one or more polynucleotide probesbased upon SEQ ID NO:1, using standard methods as described herein andknown in the art (see, for example, Ausubel (1995) at pages 6-1 to6-11).

Anti-TGF-beta binding-protein antibodies, produced as described herein,can also be used to isolate DNA sequences that encode a TGF-betabinding-protein from cDNA libraries. For example, the antibodies can beused to screen λgt11 expression libraries, or the antibodies can be usedfor immunoscreening following hybrid selection and translation (see, forexample, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al.,“Screening λ expression libraries with antibody and protein probes,” inDNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),pages 1-14 (Oxford University Press 1995)).

The sequence of a TGF-beta binding-protein cDNA or TGF-betabinding-protein genomic fragment can be determined using standardmethods. Moreover, the identification of genomic fragments containing aTGF-beta binding-protein promoter or regulatory element can be achievedusing well-established techniques, such as deletion analysis (seegenerally Ausubel (1995), supra).

As an alternative, a TGF-beta binding-protein gene can be obtained bysynthesizing DNA molecules using mutually priming long oligonucleotidesand the nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol. 21:1131, 1993;Bambot et al., PCR Methods and Applications 2:266, 1993; Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993); Holowachuk et al., PCR Methods Appl. 4:299,1995).

3. Production of TGF-Beta Binding-Protein Genes

Nucleic acid molecules encoding variant TGF-beta binding-protein genescan be obtained by screening various cDNA or genomic libraries withpolynucleotide probes having nucleotide sequences based upon SEQ IDNO:1, 5, 9, 11, 13, 15, 100, or 101 using procedures described herein.TGF-beta binding-protein gene variants can also be constructedsynthetically. For example, a nucleic acid molecule can be devised thatencodes a polypeptide having a conservative amino acid change, comparedwith the amino acid sequence of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 46,or 65. That is, variants can be obtained that contain one or more aminoacid substitutions of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 46, or 65, inwhich an alkyl amino acid is substituted for an alkyl amino acid in aTGF-beta binding-protein amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a TGF-beta binding-proteinamino acid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a TGF-beta binding-protein amino acidsequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in a TGF-beta binding-protein amino acidsequence, an acidic amino acid is substituted for an acidic amino acidin a TGF-beta binding-protein amino acid sequence, a basic amino acid issubstituted for a basic amino acid in a TGF-beta binding-protein aminoacid sequence, or a dibasic monocarboxylic amino acid is substituted fora dibasic monocarboxylic amino acid in a TGF-beta binding-protein aminoacid sequence. Among the common amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine. Inmaking such substitutions, it is important, when possible, to maintainthe cysteine backbone outlined in FIG. 1.

Conservative amino acid changes in a TGF-beta binding-protein gene canbe introduced by substituting nucleotides for the nucleotides recited inSEQ ID NO:1, 5, 9, 11, 13, 15, 100, or 101. Such “conservative aminoacid” variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel (1995) at pages8-10 to 8-22; McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)). The functional ability of such variants canbe determined using a standard method, such as the assay describedherein. Alternatively, a variant TGF-beta binding-protein polypeptidecan be identified by the ability to specifically bind anti-TGF-betabinding-protein antibodies.

Routine deletion analyses of nucleic acid molecules can be performed toobtain “functional fragments” of a nucleic acid molecule that encodes aTGF-beta binding-protein polypeptide. As an illustration, DNA moleculeshaving the nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for activity, or for theability to bind anti-TGF-beta binding-protein antibodies. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired fragment. Alternatively, particular fragments of a TGF-betabinding-protein gene can be synthesized using the polymerase chainreaction.

Standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113, 1993;Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J.Biol. Chem. 270:29270, 1995; Fukunaga et al., J. Biol. Chem. 270:25291,1995; Yamaguchi et al., Biochem. Pharmacol. 50:1295, 1995; Meisel etal., Plant Molec. Biol. 30:1, 1996.

The present invention also contemplates functional fragments of aTGF-beta binding-protein gene that have conservative amino acid changes.

A TGF-beta binding-protein variant gene can be identified on the basisof structure by determining the level of identity with nucleotide andamino acid sequences of SEQ ID NOs: 1, 5, 9, 11, 13, 15, 100, or 101 and2, 6, 10, 12, 14, 16, 46, or 65 as discussed above. An alternativeapproach to identifying a variant gene on the basis of structure is todetermine whether a nucleic acid molecule encoding a potential variantTGF-beta binding-protein gene can hybridize under stringent conditionsto a nucleic acid molecule having the nucleotide sequence of SEQ ID Nos:1, 5, 9, 11, 13, 15, 100, or 101, or a portion thereof of at least 15 or20 nucleotides in length. As an illustration of stringent hybridizationconditions, a nucleic acid molecule having a variant TGF-betabinding-protein sequence can bind with a fragment of a nucleic acidmolecule having a sequence from SEQ ID NO:1 in a buffer containing, forexample, 5×SSPE (1×SSPE=180 mM sodium chloride, 10 mM sodium phosphate,1 mM EDTA (pH 7.7), 5×Denhardt's solution (100×Denhart's=2% (w/v) bovineserum albumin, 2%. (w/v) Ficoll, 2% (w/v) polyvinylpyrrolidone) and 0.5%SDS incubated overnight at 55-60° C. Post-hybridization washes at highstringency are typically performed in 0.5×SSC (1×SSC=150 mM sodiumchloride, 15 mM trisodium citrate) or in 0.5×SSPE at 55-60° C.

Regardless of the particular nucleotide sequence of a variant TGF-betabinding-protein gene, the gene encodes a polypeptide that can becharacterized by its functional activity, or by the ability to bindspecifically to an anti-TGF-beta binding-protein antibody. Morespecifically, variant TGF-beta binding-protein genes encode polypeptideswhich exhibit at least 50%, and preferably, greater than 60, 70, 80 or90%, of the activity of polypeptides encoded by the human TGF-betabinding-protein gene described herein.

4. Production of TGF-Beta Binding-Protein in Cultured Cells

To express a TGF-beta binding-protein gene, a nucleic acid moleculeencoding the polypeptide must be operably linked to regulatory sequencesthat control transcriptional expression in an expression vector and thenintroduced into a host cell. In addition to transcriptional regulatorysequences, such as promoters and enhancers, expression vectors caninclude translational regulatory sequences and a marker gene that issuitable for selection of cells that carry the expression vector.Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence.

TGF-beta binding-proteins of the present invention are preferablyexpressed in mammalian cells. Examples of mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21; ATCC CRL 8544), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells(GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene [Hamer et al., J. Molec. Appl. Genet. 1:273, 1982], the TK promoterof Herpes virus [McKnight, Cell 31:355, 1982], the SV40 early promoter[Benoist et al., Nature 290:304, 1981], the Rous sarcoma virus promoter[Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777, 1982], thecytomegalovirus promoter [Foecking et al., Gene 45:101, 1980], and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control TGF-beta binding-proteingene expression in mammalian cells if the prokaryotic promoter isregulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol.10:4529, 1990; Kaufman et al., Nucleic Acids Res. 19:4485, 1991).

TGF-beta binding-protein genes may also be expressed in bacterial,yeast, insect, or plant cells. Suitable promoters that can be used toexpress TGF-beta binding-protein polypeptides in a prokaryotic host arewell-known to those of skill in the art and include promoters capable ofrecognizing the T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5,tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofpBR322, and the CAT promoter of the chloramphenicol acetyl transferasegene. Prokaryotic promoters have been reviewed by Glick, J. Ind.Microbiol. 1:277, 1987, Watson et al., Molecular Biology of the Gene,4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).

Preferred prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (Ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (Ed.) (IRL Press 1985)).

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995); Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995); and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

The baculovirus system provides an efficient means to introduce clonedTGF-beta binding-protein genes into insect cells. Suitable expressionvectors are based upon the Autographa californica multiple nuclearpolyhedrosis virus (AcMNPV), and contain well-known promoters such asDrosophila heat shock protein (hsp) 70 promoter, Autographa californicanuclear polyhedrosis virus immediate-early gene promoter (ie-1) and thedelayed early 39K promoter, baculovirus p10 promoter, and the Drosophilametallothionein promoter. Suitable insect host cells include cell linesderived from IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cellline, such as Sf9 (ATCC CRL 1711), Sf21 AE, and Sf21 (InvitrogenCorporation; San Diego, Calif.), as well as Drosophila Schneider-2cells. Established techniques for producing recombinant proteins inbaculovirus systems are provided by Bailey et al., “Manipulation ofBaculovirus Vectors,” in Methods in Molecular Biology, Volume 7: GeneTransfer and Expression Protocols, Murray (ed.), pages 147-168 (TheHumana Press, Inc. 1991), by Patel et al., “The baculovirus expressionsystem,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover etal. (eds.), pages 205-244 (Oxford University Press 1995), by Ausubel(1995) at pages 16-37 to 16-57, by Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995), and by Lucknow,“Insect Cell Expression Technology,” in Protein Engineering Principlesand Practice, Cleland et al. (eds.), pages 183-218 (John Wiley & Sons,Inc. 1996).

Promoters for expression in yeast include promoters from GAL1(galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase),AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.Many yeast cloning vectors have been designed and are readily available.These vectors include YIp-based vectors, such as YIp5, YRp vectors, suchas YRp17, YEp vectors such as YEp13 and YCp vectors, such as YCp19. Oneskilled in the art will appreciate that there are a wide variety ofsuitable vectors for expression in yeast cells.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. General methods of culturingplant tissues are provided, for example, by Miki et al., “Procedures forIntroducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. Preferably, the transfected cells areselected and propagated to provide recombinant host cells that comprisethe expression vector stably integrated in the host cell genome.Techniques for introducing vectors into eukaryotic cells and techniquesfor selecting such stable transformants using a dominant selectablemarker are described, for example, by Ausubel (1995) and by Murray(ed.), Gene Transfer and Expression Protocols (Humana Press 1991).Methods for introducing expression vectors into bacterial, yeast,insect, and plant cells are also provided by Ausubel (1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system is provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc., 1995).

More generally, TGF-beta binding-protein can be isolated by standardtechniques, such as affinity chromatography, size exclusionchromatography, ion exchange chromatography, HPLC and the like.Additional variations in TGF-beta binding-protein isolation andpurification can be devised by those of skill in the art. For example,anti-TGF-beta binding-protein antibodies, obtained as described below,can be used to isolate large quantities of protein by immunoaffinitypurification.

5. Production of Antibodies to TGF-Beta Binding-Proteins

The present invention provides antibodies that specifically bind tosclerostin as described herein in detail. Antibodies to TGF-betabinding-protein can be obtained, for example, using the product of anexpression vector as an antigen. Antibodies that specifically bind tosclerostin may also be prepared by using peptides derived from any oneof the sclerostin polypeptide sequences provided herein (SEQ ID NOs: 2,6, 8, 10, 12, 14, 16, 46, and 65). Particularly useful anti-TGF-betabinding-protein antibodies “bind specifically” with TGF-betabinding-protein of Sequence ID Nos. 2, 6, 8, 10, 12, 14, 16, 46, or 65but not to other TGF-beta binding-proteins such as Dan, Cerberus, SCGF,or Gremlin. Antibodies of the present invention (including fragments andderivatives thereof) may be a polyclonal or, especially a monoclonalantibody. The antibody may belong to any immunoglobulin class, and maybe for example an IgG, (including isotypes of IgG, which for humanantibodies are known in the art as IgG₁, IgG₂, IgG₃, IgG₄); IgE; IgM; orIgA antibody. An antibody may be obtained from fowl or mammals,preferably, for example, from a murine, rat, human or other primateantibody. When desired the antibody may be an internalising antibody.

Polyclonal antibodies to recombinant TGF-beta binding-protein can beprepared using methods well-known to those of skill in the art (see, forexample, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992);Williams et al., “Expression of foreign proteins in E. coli usingplasmid vectors and purification of specific polyclonal antibodies,” inDNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),page 15 (Oxford University Press 1995)). Although polyclonal antibodiesare typically raised in animals such as rats, mice, rabbits, goats, orsheep, an anti-TGF-beta binding-protein antibody of the presentinvention may also be derived from a subhuman primate antibody. Generaltechniques for raising diagnostically and therapeutically usefulantibodies in baboons may be found, for example, in Goldenberg et al.,international patent publication No. WO 91/11465 (1991), and in Losmanet al., Int. J. Cancer 46:310, 1990.

The antibody should comprise at least a variable region domain. Thevariable region domain may be of any size or amino acid composition andwill generally comprise at least one hypervariable amino acid sequenceresponsible for antigen binding embedded in a framework sequence. Ingeneral terms the variable (V) region domain may be any suitablearrangement of immunoglobulin heavy (V_(H)) and/or light (V_(L)) chainvariable domains. Thus for example the V region domain may be monomericand be a V_(H) or V_(L) domain where these are capable of independentlybinding antigen with acceptable affinity. Alternatively the V regiondomain may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L), orV_(L)-V_(L), dimers in which the V_(H) and V_(L) chains arenon-covalently associated (abbreviated hereinafter as F_(v)). Wheredesired, however, the chains may be covalently coupled either directly,for example via a disulphide bond between the two variable domains, orthrough a linker, for example a peptide linker, to form a single chaindomain (abbreviated hereinafter to as scF_(v)).

The variable region domain may be any naturally occurring variabledomain or an engineered version thereof. By engineered version is meanta variable region domain that has been created using recombinant DNAengineering techniques. Such engineered versions include those createdfor example from natural antibody variable regions by insertions,deletions or changes in or to the amino acid sequences of the naturalantibodies. Particular examples of this type include those engineeredvariable region domains containing at least one CDR and optionally oneor more framework amino acids from one antibody and the remainder of thevariable region domain from a second antibody.

The variable region domain may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example where a V_(H) domain is present in the variable regiondomain this may be linked to an immunoglobulin C_(H)1 domain or afragment thereof. Similarly a V_(L) domain may be linked to a C_(K)domain or a fragment thereof. In this way for example the antibody maybe a Fab fragment wherein the antigen binding domain contains associatedV_(H) and V_(L) domains covalently linked at their C-termini to a CH₁and C_(K) domain respectively. The CH1 domain may be extended withfurther amino acids, for example to provide a hinge region domain asfound in a Fab′ fragment, or to provide further domains, such asantibody CH2 and CH3 domains.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991;Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995); andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Antibodies for use in the invention may in general be monoclonal(prepared to by conventional immunisation and cell fusion procedures) orin the case of fragments, derived therefrom using any suitable standardchemical such as reduction or enzymatic cleavage and/or digestiontechniques, for example by treatment with pepsin. More specifically,monoclonal anti-TGF-beta binding-protein antibodies can be generatedutilizing a variety of techniques. Rodent monoclonal antibodies tospecific antigens may be obtained by methods known to those skilled inthe art (see, for example, Kohler et al., Nature 256:495, 1975; andColigan et al. (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7(John Wiley & Sons 1991) [“Coligan”]; Picksley et al., “Production ofmonoclonal antibodies against proteins expressed in E. coli,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a TGF-beta binding-protein gene product,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

In addition, an anti-TGF-beta binding-protein antibody of the presentinvention may be derived from a human monoclonal antibody. Humanmonoclonal antibodies are obtained from transgenic mice that have beenengineered to produce specific human antibodies in response to antigenicchallenge. In this technique, elements of the human heavy and lightchain locus are introduced into strains of mice derived from embryonicstem cell lines that contain targeted disruptions of the endogenousheavy chain and light chain loci. The transgenic mice can synthesizehuman antibodies specific for human antigens, and the mice can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described, for example, byGreen et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856,1994; and Taylor et al., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-TGF-beta binding-protein antibodies. Such antibody fragments can beobtained, for example, by proteolytic hydrolysis of the antibody.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. As an illustration, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing pepsin produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230,1960, Porter, Biochem. J. 73:119, 1959, Edelman et al., in Methods inEnzymology 1:422 (Academic Press 1967), and by Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Alternatively, the antibody may be a recombinant or engineered antibodyobtained by the use of recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Such DNA is known and/or is readily available from DNAlibraries including for example phage-antibody libraries (see Chiswell,D J and McCafferty, J. Tibtech. 10 80-84 (1992)) or where desired can besynthesised. Standard molecular biology and/or chemistry procedures maybe used to sequence and manipulate the DNA, for example, to introducecodons to create cysteine residues, to modify, add or delete other aminoacids or domains as desired.

One or more replicable expression vectors containing the DNA encoding avariable and/or constant region may be prepared and used to transform anappropriate cell line, e.g. a non-producing myeloma cell line, such as amouse NSO line or a bacterial, such as E. coli, in which production ofthe antibody will occur. In order to obtain efficient transcription andtranslation, the DNA sequence in each vector should include appropriateregulatory sequences, particularly a promoter and leader sequenceoperably linked to a variable domain sequence. Particular methods forproducing antibodies in this way are generally well known and routinelyused. For example, basic molecular biology procedures are described byManiatis et al (Molecular Cloning, Cold Spring Harbor Laboratory, NewYork, 1989); DNA sequencing can be performed as described in Sanger etal (Proc. Natl. Acad. Sci. USA 74: 5463, (1977)) and the AmershamInternational plc sequencing handbook; site directed mutagenesis can becarried out according to the method of Kramer et al. (Nucleic Acids Res.12, 9441, (1984)); the Anglian Biotechnology Ltd handbook; Kunkel Proc.Natl. Acad. Sci. USA 82:488-92 (1985); Kunkel et al., Methods inEnzymol. 154:367-82 (1987). Additionally, numerous publications detailtechniques suitable for the preparation of antibodies by manipulation ofDNA, creation of expression vectors, and transformation of appropriatecells, for example as reviewed by Mountain A and Adair, J R inBiotechnology and Genetic Engineering Reviews (ed. Tombs, M P, 10,Chapter 1, 1992, Intercept, Andover, UK) and in International PatentSpecification No. WO 91/09967.

In certain embodiments, the antibody according to the invention may haveone or more effector or reporter molecules attached to it and theinvention extends to such modified proteins. A reporter molecule may bea detectable moiety or label such as an enzyme, cytotoxic agent or otherreporter molecule, including a dye, radionuclide, luminescent group,fluorescent group, or biotin, or the like. The TGF-beta bindingprotein-specific immunoglobulin or fragment thereof may be radiolabeledfor diagnostic or therapeutic applications. Techniques for radiolabelingof antibodies are known in the art. See, e.g., Adams 1998 In Vivo12:11-21; Hiltunen 1993 Acta Oncol. 32:831-9. Therapeutic applicationsare described in greater detail below and may include use of theTGF-beta binding protein specific antibody (or fragment thereof) inconjunction with other therapeutic agents. The effector or reportermolecules may be attached to the antibody through any available aminoacid side-chain, terminal amino acid or, where present carbohydratefunctional group located in the antibody, provided that the attachmentor the attachment process does not adversely affect the bindingproperties and the usefulness of the molecule. Particular functionalgroups include, for example any free amino, imino, thiol, hydroxyl,carboxyl or aldehyde group. Attachment of the antibody and the effectorand/or reporter molecule(s) may be achieved via such groups and anappropriate functional group in the effector or reporter molecules. Thelinkage may be direct or indirect through spacing or bridging groups.

Effector molecules include, for example, antineoplastic agents, toxins(such as enzymatically active toxins of bacterial (such as P. aeruginosaexotoxin A) or plant origin and fragments thereof (e.g. ricin andfragments thereof; plant gelonin, bryodin from Bryonia dioica, or thelike. See, e.g., Thrush et al., 1996 Annu. Rev. Immunol. 14:49-71;Frankel et al., 1996 Cancer Res. 56:926-32); biologically activeproteins, for example enzymes; nucleic acids and fragments thereof suchas. DNA, RNA and fragments thereof; naturally occurring and syntheticpolymers (e.g., polysaccharides and polyalkylene polymers such aspoly(ethylene glycol) and derivatives thereof); radionuclides,particularly radioiodide; and chelated metals. Suitable reporter groupsinclude chelated metals, fluorescent compounds, or compounds that may bedetected by NMR or ESR spectroscopy. Particularly useful effector groupsare calichaemicin and derivatives thereof (see, for example, SouthAfrican Patent Specifications Nos. 85/8794, 88/8127 and 90/2839).

Numerous other toxins, including chemotherapeutic agents, anti-mitoticagents, antibiotics, inducers of apoptosis (or “apoptogens”, see, e.g.,Green and Reed, 1998, Science 281:1309-1312), or the like, are known tothose familiar with the art, and the examples provided herein areintended to be illustrative without limiting the scope and spirit of theinvention. Particular antineoplastic agents include cytotoxic andcytostatic agents, for example alkylating agents, such as nitrogenmustards (e.g., chlorambucil, melphalan, mechlorethamine,cyclophosphamide, or uracil mustard) and derivatives thereof,triethylenephosphoramide, triethylenethiophosphor-amide, busulphan, orcisplatin; antimetabolites, such as methotrexate, fluorouracil,floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acidor fluorocitric acid, antibiotics, such as bleomycins (e.g., bleomycinsulphate), doxorubicin, daunorubicin, mitomycins (e.g., mitomycin C),actinomycins (e.g., dactinomycin) plicamycin, calichaemicin andderivatives thereof, or esperamicin and derivatives thereof; mitoticinhibitors, such as etoposide, vincristine or vinblastine andderivatives thereof; alkaloids, such as ellipticine; polyols such astaxicin-I or taxicin-II; hormones, such as androgens (e.g.,dromostanolone or testolactone), progestins (e.g., megestrol acetate ormedroxyprogesterone acetate), estrogens (e.g., dimethylstilbestroldiphosphate, polyestradiol phosphate or estramustine phosphate) orantiestrogens (e.g., tamoxifen); anthraquinones, such as mitoxantrone,ureas, such as hydroxyurea; hydrazines, such as procarbazine; orimidazoles, such as dacarbazine.

Chelated metals include chelates of di- or tripositive metals having acoordination number from 2 to 8 inclusive. Particular examples of suchmetals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu),gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium(Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (Sc). Ingeneral the metal is preferably a radionuclide. Particular radionuclidesinclude ^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ⁵⁸Co, ⁶⁰Co, ⁶⁷Cu, ¹⁹⁵Au, ¹⁹⁹Au, ¹¹⁰Ag,²⁰³Pb, ²⁰⁶Bi, ²⁰⁷Bi, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁸⁸Y, ⁹⁰Y, ¹⁶⁰Tb, ¹⁵³Gd, and⁴⁷Sc.

The chelated metal may be for example one of the above types of metalchelated with any suitable polydentate chelating agent, for exampleacyclic or cyclic polyamines, polyethers, (e.g., crown ethers andderivatives thereof); polyamides; porphyrins; and carbocyclicderivatives. In general, the type of chelating agent will depend on themetal in use. One particularly useful group of chelating agents inconjugates according to the invention, however, comprises acyclic andcyclic polyamines, especially polyaminocarboxylic acids, for examplediethylenetriaminepentaacetic acid and derivatives thereof, andmacrocyclic amines, such as cyclic tri-aza and tetra-aza derivatives(for example, as described in International Patent Specification No. WO92/22583), and polyamides, especially desferrioxamine and derivativesthereof.

When a thiol group in the antibody is used as the point of attachmentthis may be achieved through reaction with a thiol reactive grouppresent in the effector or reporter molecule. Examples of such groupsinclude an á-halocarboxylic acid or ester, such as iodoacetamide, animide, such as maleimide, a vinyl sulphone, or a disulphide. These andother suitable linking procedures are generally and more particularlydescribed in International Patent Specifications Nos. WO 93/06231, WO92/22583, WO 90/091195, and WO 89/01476.

Assays for Selecting Molecules that Increase Bone Density

As discussed above, the present invention provides methods for selectingand/or isolating compounds that are capable of increasing bone density.For example, within one aspect of the present invention methods areprovided for determining whether a selected molecule (e.g., a candidateagent) is capable of increasing bone mineral content, comprising thesteps of (a) mixing (or contacting) a selected molecule with TGF-betabinding protein and a selected member of the TGF-beta family ofproteins, (b) determining whether the selected molecule stimulatessignaling by the TGF-beta family of proteins, or inhibits the binding ofthe TGF-beta binding protein to at least one member of the TGF-betafamily of proteins. Within certain embodiments, the molecule enhancesthe ability of TGF-beta to function as a positive regulator ofmesenchymal cell differentiation.

Within other aspects of the invention, methods are provided fordetermining whether a selected molecule (candidate agent) is capable ofincreasing bone mineral content, comprising the steps of (a) exposing(contacting, mixing, combining) a selected molecule to cells whichexpress TGF-beta binding-protein and (b) determining whether theexpression (or activity) of TGF-beta binding-protein in the exposedcells decreases, or whether an activity of the TGF-beta binding proteindecreases, and therefrom determining whether the compound is capable ofincreasing bone mineral content. Within one embodiment, the cells areselected from the group consisting of the spontaneously transformed oruntransformed normal human bone from bone biopsies and rat parietal boneosteoblasts. Methods for detecting the level of expression of a TGF-betabinding protein may be accomplished in a wide variety of assay formatsknown in the art and described herein. Immunoassays may be used fordetecting and quantifying the expression of a TGF-beta binding proteinand include, for example, Countercurrent Immuno-Electrophoresis (CIEP),radioimmunoassays, radioimmunoprecipitations, Enzyme-LinkedImmuno-Sorbent Assays (ELISA), immunoblot assays such as dot blot assaysand Western blots, inhibition or competition assays, and sandwich assays(see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: ALaboratory Manual, supra). Such immunoassays may use an antibody that isspecific for a TGF-beta binding protein such as the anti-sclerostinantibodies described herein, or may use an antibody that is specific fora reporter molecule that is attached to the TGF-beta binding protein.The level of polypeptide expression may also be determined byquantifying the amount of TGF-beta binding protein that binds to aTGF-beta binding protein ligand. By way of example, binding ofsclerostin in a sample to a BMP may be detected by surface plasmonresonance (SPR). Alternatively, the level of expression of mRNA encodingthe specific TGF-beta binding protein may be quantified.

Representative embodiments of such assays are provided below in Examples5 and 6. Briefly, a family member of the TGF-beta super-family or aTGF-beta binding protein is first bound to a solid phase, followed byaddition of a candidate molecule. A labeled family member of theTGF-beta super-family or a TGF-beta binding protein is then added to theassay (i.e., the labeled polypeptide is the ligand for whicheverpolypeptide was bound to the solid phase), the solid phase washed, andthe quantity of bound or labeled TGF-beta super-family member orTGF-beta binding protein on the solid support determined. Moleculeswhich are suitable for use in increasing bone mineral content asdescribed herein are those molecules which decrease the binding ofTGF-beta binding protein to a member or members of the TGF-betasuper-family in a statistically significant manner. Obviously, assayssuitable for use within the present invention should not be limited tothe embodiments described within Examples 2 and 3. In particular,numerous parameters may be altered, such as by binding TGF-beta to asolid phase, or by elimination of a solid phase entirely.

Within other aspects of the invention, methods are provided fordetermining whether a selected molecule is capable of increasing bonemineral content, comprising the steps of (a) exposing (contacting,mixing, combining) a selected molecule (candidate agent) to cells whichexpress TGF-beta and (b) determining whether the activity of TGF-betafrom said exposed cells is altered, and therefrom determining whetherthe compound is capable of increasing bone mineral content. Similar tothe methods described herein, a wide variety of methods may be utilizedto assess the changes of TGF-beta binding-protein expression due to aselected test compound. In one embodiment of the invention, thecandidate agent is an antibody that binds to the TGF-beta bindingprotein sclerostin disclosed herein.

In a preferred embodiment of the invention, a method is provided foridentifying an antibody that modulates a TGF-beta signaling pathwaycomprising contacting an antibody that specifically binds to a SOSTpolypeptide with a SOST peptide, including but not limited to thepeptides disclosed herein, under conditions and for a time sufficient topermit formation of an antibody plus (+) SOST (antibody/SOST) complexand then detecting the level (e.g., quantifying the amount) of theSOST/antibody complex to determine the presence of an antibody thatmodulates a TGF-beta signaling pathway. The method may be performedusing SPR or any number of different immunoassays known in the art anddisclosed herein, including an ELISA, immunoblot, or the like. ATGF-beta signaling pathway includes a signaling pathway by which a BMPbinds to a type I and a type II receptor on a cell to stimulate orinduce the pathway that modulates bone mineral content. In certainpreferred embodiments of the invention, an antibody that specificallybinds to SOST stimulates or enhances the pathway for increasing bonemineral content. Such an antibody may be identified using the methodsdisclosed herein to detect binding of an antibody to SOST specificpeptides.

The subject invention methods may also be used for identifyingantibodies that impair, inhibit (including competitively inhibit), orprevent binding of a BMP to a SOST polypeptide by detecting whether anantibody binds to SOST peptides that are located in regions or portionsof regions on SOST to which a BMP binds, such as peptides at the aminoterminal end of SOST and peptides that include amino terminal amino acidresidues and a portion of the core region (docking core) of SOST (e.g.,SEQ ID NOs:47-64, 66-73, and 92-95). The methods of the presentinvention may also be used to identify an antibody that impairs,prevents, or inhibits, formation of SOST homodimers. Such an antibodythat binds specifically to SOST may be identified by detecting bindingof the antibody to peptides that are derived from the core or thecarboxy terminal region of SOST (e.g., SEQ ID NOs: 74-91 and 96-99).

Within another embodiment of the present invention, methods are providedfor determining whether a selected molecule is capable of increasingbone mineral content, comprising the steps of (a) mixing or contacting aselected molecule (candidate agent) with a TGF-beta-binding-protein anda selected member of the TGF-beta family of proteins, (b) determiningwhether the selected molecule up-regulates the signaling of the TGF-betafamily of proteins, or inhibits the binding of the TGF-betabinding-protein to the TGF-beta family of proteins. Within certainembodiments, the molecule enhances the ability of TGF-beta to functionas a positive regulator of mesenchymal cell differentiation.

Similar to the above described methods, a wide variety of methods may beutilized to assess stimulation of TGF-beta due to a selected testcompound. One such representative method is provided below in Example 6(see also Durham et al., Endo. 136:1374-1380.

Within yet other aspects of the present invention, methods are providedfor determining whether a selected molecule (candidate agent) is capableof increasing bone mineral content, comprising the step of determiningwhether a selected molecule inhibits the binding of TGF-betabinding-protein to bone, or an analogue thereof. As utilized herein, itshould be understood that bone or analogues thereof refers tohydroxyapatite, or a surface composed of a powdered form of bone,crushed bone or intact bone. Similar to the above described methods, awide variety of methods may be utilized to assess the inhibition ofTGF-beta binding-protein localization to bone matrix. One suchrepresentative method is provided below in Example 7 (see also Nicolaset al., Calcif. Tissue Int. 47:206-12 (1995)).

In one embodiment of the invention, an antibody or antigen-bindingfragment thereof that specifically binds to a sclerostin polypeptide iscapable of competitively inhibiting binding of a TGF-beta family memberto the sclerostin polypeptide. The capability of the antibody orantibody fragment to impair or blocking binding of a TGF-beta familymember, such as a BMP, to sclerostin may be determined according to anyof the methods described herein. The antibody or fragment thereof thatspecifically binds to sclerostin may impair, block, or prevent bindingof a TGF-beta family member to sclerostin by impairing sclerostinhomodimer formation. An antibody that specifically binds to sclerostinmay also be used to identify an activity of sclerostin by inhibiting orimpairing sclerostin from binding to a BMP. Alternatively, the antibodyor fragment thereof may be incorporated in a cell-based assay or in ananimal model in which sclerostin has a defined activity to determinewhether the antibody alters (increases or decreases in a statisticallysignificant manner) that activity. An antibody or fragment thereof thatspecifically binds to sclerostin may be used to examine the effect ofsuch an antibody in a signal transduction pathway and thereby modulate(stimulate or inhibit) the signaling pathway. Preferably, binding of anantibody to SOST results in a stimulation or induction of a signalingpathway.

While the methods recited herein may refer to the analysis of anindividual test molecule, that the present invention should not be solimited. In particular, the selected molecule may be contained within amixture of compounds. Hence, the recited methods may further comprisethe step of isolating a molecule that inhibits the binding of TGF-betabinding-protein to a TGF-beta family member.

Candidate Molecules

A wide variety of molecules may be assayed for their ability to inhibitthe binding of TGF-beta binding-protein to a TGF-beta family member.Representative examples discussed in more detail below include organicmolecules (e.g., organic small molecules), proteins or peptides, andnucleic acid molecules. Although it should be evident from thediscussion below that the candidate molecules described herein may beutilized in the assays described herein, it should also be readilyapparent that such molecules can also be utilized in a variety ofdiagnostic and therapeutic settins.

1. Organic Molecules

Numerous organic small molecules may be assayed for their ability toinhibit the binding of TGF-beta binding-protein to a TGF-beta familymember. For example, within one embodiment of the invention suitableorganic molecules may be selected from either a chemical library,wherein chemicals are assayed individually, or from combinatorialchemical libraries where multiple compounds are assayed at once, thendeconvoluted to determine and isolate the most active compounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties,”U.S. Pat. No. 5,463,564; Armstrong, R. W., “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses,” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use,” WO 95/24186; Baldwin, J. J. etal., “Combinatorial dihydrobenzopyran library,” WO 95/30642; Brenner,S., “New kit for preparing combinatorial libraries,” WO 95/16918;Chenera, B. et al., “Preparation of library of resin-bound aromaticcarbocyclic compounds,” WO 95/16712; Ellman, J. A., “Solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support,”U.S. Pat. No. 5,288,514; Felder, E. et al., “Novel combinatorialcompound libraries,” WO 95/16209; Lerner, R. et al., “Encodedcombinatorial chemical libraries,” WO 93/20242; Pavia, M. R. et al., “Amethod for preparing and selecting pharmaceutically useful non-peptidecompounds from a structurally diverse universal library,” WO 95/04277;Summerton, J. E. and D. D. Weller, “Morpholino-subunit combinatoriallibrary and method,” U.S. Pat. No. 5,506,337; Holmes, C., “Methods forthe Solid Phase Synthesis of Thiazolidinones, Metathiazanones, andDerivatives thereof,” WO 96/00148; Phillips, G. B. and G. P. Wei,“Solid-phase Synthesis of Benzimidazoles,” Tet. Letters 37:4887-90,1996; Ruhland, B. et al., “Solid-supported Combinatorial Synthesis ofStructurally Diverse β-Lactams,” J. Amer. Chem. Soc. 111:253-4, 1996;Look, G. C. et. al., “The Identification of Cyclooxygenase-1 Inhibitorsfrom 4-Thiazolidinone Combinatorial Libraries,” Bioorg and Med. Chem.Letters 6:707-12, 1996.

2. Proteins and Peptides

A wide range of proteins and peptides may likewise be utilized ascandidate molecules for inhibitors of the binding of TGF-betabinding-protein to a TGF-beta family member.

a. Combinatorial Peptide Libraries

Peptide molecules which are putative inhibitors of the binding ofTGF-beta binding-protein to a TGF-beta family member may be obtainedthrough the screening of combinatorial peptide libraries. Such librariesmay either be prepared by one of skill in the art (see e.g., U.S. Pat.Nos. 4,528,266 and 4,359,535, and Patent Cooperation Treaty PublicationNos. WO 92/15679, WO 92/15677, WO 90/07862, WO 90/02809, or purchasedfrom commercially available sources (e.g., New England Biolabs Ph.D.™Phage Display Peptide Library Kit).

b. Antibodies

The present invention provides antibodies that specifically bind to asclerostin polypeptide methods for using such antibodies. The presentinvention also provides sclerostin polypeptide immunogens that may beused for generation and analysis of these antibodies. The antibodies maybe useful to block or impair binding of a sclerostin polypeptide, whichis a TGF-beta binding protein, to a ligand, particularly a bonemorphogenic protein, and may also block or impair binding of thesclerostin polypeptide to one or more other ligands.

A molecule such as an antibody that inhibits the binding of the TGF-betabinding protein to one or more members of the TGF-beta family ofproteins, including one or more bone morphogenic proteins (BMPs), shouldbe understood to refer to, for example, a molecule that allows theactivation of a TGF-beta family member or BMP, or allows binding ofTGF-beta family members including one or more BMPs to their respectivereceptors by removing or preventing the TGF-beta member from binding tothe TGF-binding-protein.

The present invention also provides peptide and polypeptide immunogensthat may be used to generate and/or identify antibodies or fragmentsthereof that are capable of inhibiting, preventing, or impairing bindingof the TGF-beta binding protein sclerostin to one or more BMPs. Thepresent invention also provides peptide and polypeptide immunogens thatmay be used to generate and/or identify antibodies or fragments thereofthat are capable of inhibiting, preventing, or impairing (e.g.,decreasing in a statistically significant manner) the formation ofsclerostin homodimers. The antibodies of the present invention areuseful for increasing the mineral content and mineral density of bone,thereby ameliorating numerous conditions that result in the loss of bonemineral content, including for example, disease, genetic predisposition,accidents that result in the lack of use of bone (e.g., due tofracture), therapeutics that effect bone resorption or that kill boneforming cells, and normal aging.

Polypeptides or peptides useful for immunization and/or analysis ofsclerostin-specific antibodies may also be selected by analyzing theprimary, secondary, and tertiary structure of a TGF-beta binding proteinaccording to methods known to those skilled in the art and describedherein, in order to determine amino acid sequences more likely togenerate an antigenic response in a host animal. See, e.g., Novotny,Mol. Immunol. 28:201-207 (1991); Berzofsky, Science 229:932-40 (1985)).Modeling and x-ray crystallography data may also be used to predictand/or identify which portions or regions of a TGF-beta binding proteininteract with which portions of a TGF-beta binding protein ligand, suchas a BMP. TGF-beta binding protein peptide immunogens may be designedand prepared that include amino acid sequences within or surrounding theportions or regions of interaction. These antibodies may be useful toblock or impair binding of the TGF-beta binding protein to the sameligand and may also block or impair binding of the TGF-beta bindingprotein to one or more other ligands.

Antibodies or antigen binding fragments thereof contemplated by thepresent invention include antibodies that are capable of specificallybinding to sclerostin and competitively inhibiting binding of a TGF-betapolypeptide, such as a BMP, to sclerostin. For example, the antibodiescontemplated by the present invention competitively inhibit binding ofthe sclerostin polypeptide to the BMP Type I receptor site on a BMP, orto the BMP Type II receptor binding site, or may competitively inhibitbinding of sclerostin to both the Type I and Type II receptor bindingsites on a BMP. Without wishing to be bound by theory, when ananti-sclerostin antibody competitively inhibits binding of the Type Iand/or Type II binding sites of the BMP polypeptide to sclerostin, thusblocking the antagonistic activity of sclerostin, the receptor bindingsites on BMP are available to bind to the Type I and Type II receptors,thereby increasing bone mineralization. The binding interaction betweena TGF-beta binding protein such as sclerostin and a TGF-beta polypeptidesuch as a BMP generally occurs when each of the ligand pairs forms ahomodimer. Therefore instead of or in addition to using an antibodyspecific for sclerostin to block, impair, or prevent binding ofsclerostin to a BMP by competitively inhibiting binding of sclerostin toBMP, a sclerostin specific antibody may be used to block or impairsclerostin homodimer formation.

By way of example, one dimer of human Noggin, which is a BMP antagonistthat has the ability to bind a BMP with high affinity (Zimmerman et al.,supra), was isolated in complex with one dimer of human BMP-7 andanalyzed by multiwavelength anomalous diffraction (MAD) (Groppe et al.,Nature 420:636-42 (2002)). As discussed herein, this study revealed thatNoggin dimer may efficiently block all the receptor binding sites (twotype I and two type II receptor binding sites) on a BMP dimer. Thelocation of the amino acids of Noggin that contact BMP-7 may be usefulin modeling the interaction between other TGF-beta binding proteins,such as sclerostin (SOST), and BMPs, and thus aiding the design ofpeptides that may be used as immunogens to generate antibodies thatblock or impair such an interaction.

In one embodiment of the present invention, an antibody, or anantigen-binding fragment thereof, that binds specifically to a SOSTpolypeptide competitively inhibits binding of the SOST polypeptide to atleast one or both of a bone morphogenic protein (BMP) Type I Receptorbinding site and a BMP Type II Receptor binding site that are located ona BMP. The epitopes on SOST to which these antibodies bind may includeor be included within contiguous amino acid sequences that are locatedat the N-terminus of the SOST polypeptide (amino acids at aboutposition)-56 of SEQ ID NO:46). The polypeptides may also include a shortlinker peptide sequence that connects the N-terminal region to the coreregion, for example, polypeptides as provided in SEQ ID NO:92 (human)and SEQ ID NO:93 (rat). Shorter representative N-terminus peptidesequences of human SOST (e.g., SEQ ID NO:46) include SEQ ID NOS:47-51,and representative rat SOST (e.g., SEQ ID NO:65) peptide sequencesinclude SEQ ID NOS:57-60.

Antibodies that specifically bind to a SOST polypeptide and block orcompetitively inhibit binding of the SOST polypeptide to a BMP, forexample, by blocking or inhibiting binding to amino acids of a BMPcorresponding to one or more of the Type I and Type II receptor bindingsites may also specifically bind to peptides that comprise an amino acidsequence corresponding to the core region of SOST (amino acids at aboutpositions 57-146 of SEQ ID NO:46). Polypeptides that include the coreregion may also include additional amino acids extending at either orboth the N-terminus and C-terminus, for example, to include cysteineresidues that may be useful for conjugating the polypeptide to a carriermolecule. Representative core polypeptides of human and rat SOST, forexample, comprise the amino acid sequences set forth in SEQ ID NO:94 andSEQ ID NO:95, respectively. Such antibodies may also bind shorterpolypeptide sequences. Representative human SOST core peptide sequencesare provided in SEQ ID NOs:66-69 and representative rat SOST coresequences are provided in SEQ ID NOs:70-73.

In another embodiment, antibodies that specifically bind to a SOSTpolypeptide impair (inhibit, prevent, or block, e.g., decrease in astatistically significant manner) formation of a SOST homodimer. Becausethe interaction between SOST and a BMP may involve a homodimer of SOSTand a homodimer of the BMP, an antibody that prevents or impairshomodimer formation of SOST may thereby alter bone mineral density,preferably increasing bone mineral density. In one embodiment,antibodies that bind to the core region of SOST prevent homodimerformation. Such antibodies may also bind to peptides that comprisecontiguous amino acid sequences corresponding the core region, forexample, SEQ ID NOs: 74, 75, and 98 (human SOST) and SEQ ID NOs:76 and99 (rat SOST). Antibodies that bind to an epitope located on theC-terminal region of a SOST polypeptide (at about amino acid positions147-190 of either SEQ ID NO:46 or 65) may also impair homodimerformation. Representative C-terminal polypeptides of human and rat SOST,for example, comprise the amino acid sequences set forth in SEQ ID NO:96and SEQ ID NO:97, respectively. Such antibodies may also bind shorterpolypeptide sequences. Representative human SOST C-terminal peptidesequences are provided in SEQ ID NOs:78-81 and representative rat SOSTC-terminal sequences are provided in SEQ ID NOs:86-88.

The SOST polypeptides and peptides disclosed herein to which antibodiesmay specifically bind are useful as immunogens. These immunogens of thepresent invention may be used for immunizing an animal to generate ahumoral immune response that results in production of antibodies thatspecifically bind to a Type I or Type II receptor binding site or bothlocated on a BMP include peptides derived from the N-terminal region ofSOST or that may prevent SOST homodimer formation.

Such SOST polypeptides and peptides that are useful as immunogens mayalso be used in methods for screening samples containing antibodies, forexample, samples of purified antibodies, antisera, or cell culturesupernatants or any other biological sample that may contain one or moreantibodies specific for SOST. These peptides may also be used in methodsfor identifying and selecting from a biological sample one or more Bcells that are producing an antibody that specifically binds to SOST(e.g., plaque forming assays and the like). The B cells may then be usedas source of a SOST specific antibody-encoding polynucleotide that canbe cloned and/or modified by recombinant molecular biology techniquesknown in the art and described herein.

A “biological sample” as used herein refers in certain embodiments to asample containing at least one antibody specific for a SOST polypeptide,and a biological sample may be provided by obtaining a blood sample,biopsy specimen, tissue explant, organ culture, or any other tissue orcell preparation from a subject or a biological source. A sample mayfurther refer to a tissue or cell preparation in which the morphologicalintegrity or physical state has been disrupted, for example, bydissection, dissociation, solubilization, fractionation, homogenization,biochemical or chemical extraction, pulverization, lyophilization,sonication, or any other means for processing a sample derived from asubject or biological source. The subject or biological source may be ahuman or non-human animal, a primary cell culture (e.g., B cellsimmunized in vitro), or culture adapted cell line including but notlimited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid celllines, differentiated or differentiable cell lines, transformed celllines, and the like.

SOST peptide immunogens may also be prepared by synthesizing a series ofpeptides that, in total, represent the entire polypeptide sequence of aSOST polypeptide and that each have a portion of the SOST amino acidsequence in common with another peptide in the series. This overlappingportion would preferably be at least four amino acids, and morepreferably 5, 6, 7, 8, 9, or 10 amino acids. Each peptide may be used toimmunize an animal, the sera collected from the animal, and tested in anassay to identify which animal is producing antibodies that impair orblock binding of SOST to a TGF-beta protein. Antibodies are thenprepared from such identified immunized animals according to methodsknown in the art and described herein.

Antibodies which inhibit the binding of TGF-beta binding-protein to aTGF-beta family member may readily be prepared given the disclosureprovided herein. Particularly useful are anti-TGF-beta binding-proteinantibodies that “specifically bind” TGF-beta binding-protein of SEQ IDNOs: 2, 6, 8, 10, 12, 14, 16, 46, or 65, but not to other TGF-betabinding-proteins such as Dan, Cerberus, SCGF, or Gremlin. Within thecontext of the present invention, antibodies are understood to includemonoclonal antibodies, polyclonal antibodies, single chain, chimeric,CDR-grafted immunoglobulings, anti-idiotypic antibodies, and antibodyfragments thereof (e.g., Fab, Fd, Fab′, and F(ab′)₂, F_(v), variableregions, or complementarity determining regions). As discussed above,antibodies are understood to be specific against TGF-betabinding-protein, or against a specific TGF-beta family member, if theybind with a K_(a) of greater than or equal to 10⁷ M⁻¹, preferablygreater than or equal to 10⁸ M⁻¹, and do not bind to other TGF-betabinding-proteins, or bind with a K_(a) of less than or equal to 10⁶ M⁻¹.Affinity of an antibody for its cognate antigen is also commonlyexpressed as a dissociation constant K_(D), and an anti-SOST is antibodyspecifically binds to a TGF-beta family member if it binds with a K_(D)of less than or equal to about 10⁻⁵ M, more preferably less than orequal to about 10⁻⁶ M, still more preferably less than or equal to 10⁻⁷M, and still more preferably less than or equal to 10⁻⁸ M. Furthermore,antibodies of the present invention preferably block, impair, or inhibit(e.g., decrease with statistical significance) the binding of TGF-betabinding-protein to a TGF-beta family member. The affinity of amonoclonal antibody or binding partner, as well as inhibition of bindingcan be readily determined by one of ordinary skill in the art (seeScatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949). Affinity may also bedetermined by surface plasmon resonance (SPR; BIAcore, Biosensor,Piscataway, N.J.). For surface plasmon resonance, target molecules areimmobilized on a solid phase and exposed to ligands in a mobile phaserunning along a flow cell. If ligand binding to the immobilized targetoccurs, the local refractive index changes, leading to a change in SPRangle, which can be monitored in real time by detecting changes in theintensity of the reflected light. The rates of change of the SPR signalcan be analyzed to yield apparent rate constants for the association anddissociation phases of the binding reaction. The ratio of these valuesgives the apparent equilibrium constant (affinity) (see, e.g., Wolff etal., Cancer Res. 53:2560-65 (1993)).

An antibody according to the present invention may belong to anyimmunoglobulin class, for example IgG, IgE, IgM, IgD, or IgA, and may beany one of the different isotypes that may comprise a class (such asIgG1, IgG2, IgG3, and IgG4 of the human IgG class). It may be obtainedfrom or derived from an animal, for example, fowl (e.g., chicken) andmammals, which includes but is not limited to a mouse, rat, hamster,rabbit, or other rodent, a cow, horse, sheep, goat, camel, human, orother primate. The antibody may be an internalising antibody.

Methods well known in the art may be used to generate antibodies,polyclonal antisera, or monoclonal antibodies that are specific for aTGF-beta binding protein such as SOST. Antibodies also may be producedas genetically engineered immunoglobulins (Ig) or Ig fragments designedto have desirable properties. For example, by way of illustration andnot limitation, antibodies may include a recombinant IgG that is achimeric fusion protein having at least one variable (V) region domainfrom a first mammalian species and at least one constant region domainfrom a second, distinct mammalian species. Most commonly, a chimericantibody has murine variable region sequences and human constant regionsequences. Such a murine/human chimeric immunoglobulin may be“humanized” by grafting the complementarity determining regions (CDRs)derived from a murine antibody, which confer binding specificity for anantigen, into human-derived V region framework regions and human-derivedconstant regions. Fragments of these molecules may be generated byproteolytic digestion, or optionally, by proteolytic digestion followedby mild reduction of disulfide bonds and alkylation. Alternatively, suchfragments may also be generated by recombinant genetic engineeringtechniques.

Certain preferred antibodies are those antibodies that inhibit or blocka TGF-beta binding protein activity within an in vitro assay, asdescribed herein. Binding properties of an antibody to a TGF-betabinding protein may generally be assessed using immunodetection methodsincluding, for example, an enzyme-linked immunosorbent assay (ELISA),immunoprecipitation, immunoblotting, countercurrentimmunoelectrophoresis, radioimmunoassays, dot blot assays, inhibition orcompetition assays, and the like, which may be readily performed bythose having ordinary skill in the art (see, e.g., U.S. Pat. Nos.4,376,110 and 4,486,530; Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988)).

An immunogen may be comprised of cells expressing a TGF-beta bindingprotein, purified or partially purified TGF-beta binding polypeptides,or variants or fragments (i.e., peptides) thereof, or peptides derivedfrom a TGF-beta binding protein. Such peptides may be generated byproteolytic cleavage of a larger polypeptide, by recombinant molecularmethodologies, or may be chemically synthesized. For instance, nucleicacid sequences encoding TGF-beta binding proteins are provided herein,such that those skilled in the art may routinely prepare TGF-betabinding proteins for use as immunogens. Peptides may be chemicallysynthesized by methods as described herein and known in the art.Alternatively, peptides may be generated by proteolytic cleavage of aTGF-beta binding protein, and individual peptides isolated by methodsknown in the art such as polyacrylamide gel electrophoresis or anynumber of liquid chromatography or other separation methods. Peptidesuseful as immunogens typically may have an amino acid sequence of atleast 4 or 5 consecutive amino acids from a TGF-beta binding proteinamino acid sequence such as those described herein, and preferably haveat least 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 19 or 20 consecutiveamino acids of a TGF-beta binding protein. Certain other preferredpeptide immunogens comprise at least 6 but no more than 12 or moreconsecutive amino acids of a TGF-beta binding protein sequence, andother preferred peptide immunogens comprise at least 21 but no more than50 consecutive amino acids of a SOST polypeptide. Other preferredpeptide immunogens comprise 21-25, 26-30, 31-35, 36-40, 41-50, or anywhole integer number of amino acids between and including 21 and 100consecutive amino acids, and between 100 and 190 consecutive amino acidsof a TGF-beta binding protein sequence.

As disclosed herein, polyclonal antibodies may be readily generated byone of ordinary skill in the art from a variety of warm-blooded animalssuch as horses, cows, various fowl, rabbits, mice, sheep, goats,baboons, or rats. Typically, the TGF-beta binding-protein or uniquepeptide thereof of 13-20 amino acids or as described herein (preferablyconjugated to keyhole limpet hemocyanin by cross-linking withglutaraldehyde) is used to immunize the animal through intraperitoneal,intramuscular, intraocular, intradermal, or subcutaneous injections,along with an adjuvant such as Freund's complete or incomplete adjuvant,or the Ribi Adjuvant System (Corixa Corporation, Seattle, Wash.). Seealso, e.g., Harlow et al., supra. In general, after the first injection,animals receive one or more booster immunizations according to apreferred schedule that may vary according to, inter alia, the antigen,the adjuvant (if any), and/or the particular animal species. The immuneresponse may be monitored by periodically bleeding the animal andpreparing and analyzing sera in an immunoassay, such as an ELISA orOuchterlony diffusion assay, or the like, to determine the specificantibody titer. Particularly preferred polyclonal antisera will give adetectable signal on one of these assays, such as an ELISA, that ispreferably at least three times greater than background. Once the titerof the animal has reached a plateau in terms of its reactivity to theprotein, larger quantities of antisera may be readily obtained either byweekly bleedings, or by exsanguinating the animal.

Polyclonal antibodies that bind specifically to the TGF-beta bindingprotein or peptide may then be purified from such antisera, for example,by affinity chromatography using protein A. Alternatively, affinitychromatography may be performed wherein the TGF-beta binding protein orpeptide or an antibody specific for an Ig constant region of theparticular immunized animal species is immobilized on a suitable solidsupport.

Antibodies for use in the invention include monoclonal antibodies thatare prepared by conventional immunization and cell fusion procedures asdescribed herein an known in the art. Monoclonal antibodies may bereadily generated using conventional techniques (see, e.g., Kohler etal., Nature 256:495, 1975; Coligan et al. (eds.), Current Protocols inImmunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991) [“Coligan”]; U.S.Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which areincorporated herein by reference; see also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988, which are also incorporated herein by reference; Picksley et al.,“Production of monoclonal antibodies against proteins expressed in E.coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), page 93 (Oxford University Press 1995)). Antibody fragments maybe derived therefrom using any suitable standard technique such asproteolytic digestion, or optionally, by proteolytic digestion (forexample, using papain or pepsin) followed by mild reduction of disulfidebonds and alkylation. Alternatively, such fragments may also begenerated by recombinant genetic engineering techniques.

Briefly, within one embodiment a subject animal such as a rat or mouseor hamster is immunized with TGF-beta binding-protein or a portion of aregion thereof, including peptides within a region, as described herein.The protein may be admixed with an adjuvant such as Freund's complete orincomplete adjuvant or Ribi adjuvant in order to increase the resultantimmune response. Between one and three weeks after the initialimmunization the animal may be reimmunized with another boosterimmunization, and tested for reactivity to the protein using assaysdescribed herein. Once the animal has reached a plateau in itsreactivity to the injected protein, it is sacrificed, and organs whichcontain large numbers of B cells such as the spleen and lymph nodes areharvested. The harvested spleen and/or lymph node cell suspensions arefused with a suitable myeloma cell that is drug-sensitized in order tocreate a “hybridoma” which secretes monoclonal antibody. Suitablemyeloma lines include, for example, NS-0, SP20, NS-1 (ATCC No. TIB 18),and P3X63-Ag 8.653 (ATCC No. CRL 1580).

The lymphoid (e.g., spleen) cells and the myeloma cells may be combinedfor a few minutes with a membrane fusion-promoting agent, such aspolyethylene glycol or a nonionic detergent, and then plated at lowdensity on a selective medium that supports the growth of hybridomacells but not unfused myeloma cells. Following the fusion, the cells maybe placed into culture plates containing a suitable medium, such as RPMI1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences,Lenexa, Kans.), as well as additional ingredients, such as fetal bovineserum (FBS, i.e., from Hyclone, Logan, Utah, or JRH Biosciences).Additionally, the medium should contain a reagent which selectivelyallows for the growth of fused spleen and myeloma cells such as HAT(hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St.Louis, Mo.). After about seven days, the resulting fused cells orhybridomas may be screened in order to determine the presence ofantibodies which are reactive with TGF-beta binding-protein (dependingon the antigen used), and which block, impair, or inhibit the binding ofTGF-beta binding-protein to a TGF-beta family member. Hybridomas thatproduce monoclonal antibodies that specifically bind to sclerostin or avariant thereof are preferred.

A wide variety of assays may be utilized to determine the presence ofantibodies which are reactive against the proteins of the presentinvention, including for example countercurrent immuno-electrophoresis,radioimmunoassays, radioimmunoprecipitations, enzyme-linkedimmuno-sorbent assays (ELISA), dot blot assays, western blots,immunoprecipitation, inhibition or competition assays, and sandwichassays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies:A Laboratory Manual, Harlow and Lane (eds.), Cold Spring HarborLaboratory Press, 1988). The hybridomas are cloned, for example, bylimited dilution cloning or by soft agar plaque isolation, andreassayed. Thus, a hybridoma producing antibodies reactive against thedesired protein may be isolated.

The monoclonal antibodies from the hybridoma cultures may be isolatedfrom the supernatants of hybridoma cultures. An alternative method forproduction of a murine monoclonal antibody is to inject the hybridomacells into the peritoneal cavity of a syngeneic mouse, for example, amouse that has been treated (e.g., pristane-primed) to promote formationof ascites fluid containing the monoclonal antibody. Monoclonalantibodies can be isolated and purified by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography (see, for example, Coligan at pages2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., “Purification ofImmunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies may bepurified by affinity chromatography using an appropriate ligand selectedbased on particular properties of the antibody (e.g., heavy or lightchain isotype, binding specificity, etc.). Examples of a suitableligand, immobilized on a solid support, include Protein A, Protein G, ananti-constant region (light chain or heavy chain) antibody, ananti-idiotype antibody, and a TGF-beta binding protein, or fragment orvariant thereof.

In addition, an anti-TGF-beta binding-protein antibody of the presentinvention may be a human monoclonal antibody. Human monoclonalantibodies may be generated by any number of techniques with which thosehaving ordinary skill in the art will be familiar. Such methods include,but are not limited to, Epstein Barr Virus (EBV) transformation of humanperipheral blood cells (e.g., containing B lymphocytes), in vitroimmunization of human B cells, fusion of spleen cells from immunizedtransgenic mice carrying inserted human immunoglobulin genes, isolationfrom human immunoglobulin V region phage libraries, or other proceduresas known in the art and based on the disclosure herein. For example,human monoclonal antibodies may be obtained from transgenic mice thathave been engineered to produce specific human antibodies in response toantigenic challenge. Methods for obtaining human antibodies fromtransgenic mice are described, for example, by Green et al., NatureGenet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al.,Int. Immun. 6:579, 1994; U.S. Pat. No. 5,877,397; Bruggemann et al.,1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N.Y.Acad. Sci. 764:525-35. In this technique, elements of the human heavyand light chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. (See also Bruggemann etal., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, humanimmunoglobulin transgenes may be mini-gene constructs, or transloci onyeast artificial chromosomes, which undergo B cell-specific DNArearrangement and hypermutation in the mouse lymphoid tissue. Humanmonoclonal antibodies may be obtained by immunizing the transgenic mice,which may then produce human antibodies specific for the antigen.Lymphoid cells of the immunized transgenic mice can be used to producehuman antibody-secreting hybridomas according to the methods describedherein. Polyclonal sera containing human antibodies may also be obtainedfrom the blood of the immunized animals.

Another method for generating human TGF-beta binding protein specificmonoclonal antibodies includes immortalizing human peripheral bloodcells by EBV transformation. See, e.g., U.S. Pat. No. 4,464,456. Such animmortalized B cell line (or lymphoblastoid cell line) producing amonoclonal antibody that specifically binds to a TGF-beta bindingprotein (or a variant or fragment thereof) can be identified byimmunodetection methods as provided herein, for example, an ELISA, andthen isolated by standard cloning techniques. The stability of thelymphoblastoid cell line producing an anti-TGF-beta binding proteinantibody may be improved by fusing the transformed cell line with amurine myeloma to produce a mouse-human hybrid cell line according tomethods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89(1989)). Still another method to generate human monoclonal antibodies isin vitro immunization, which includes priming human splenic B cells withantigen, followed by fusion of primed B cells with a heterohybrid fusionpartner. See, e.g., Boerner et al., 1991. J. Immunol. 147:86-95.

In certain embodiments, a B cell that is producing an anti-SOST antibodyis selected and the light chain and heavy chain variable regions arecloned from the B cell according to molecular biology techniques knownin the art (WO 92/02551; U.S. Pat. No. 5,627,052; Babcook et al., Proc.Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. PreferablyB cells from an immunized animal are isolated from the spleen, lymphnode, or peripheral blood sample by selecting a cell that is producingan antibody that specifically binds to SOST. B cells may also beisolated from humans, for example, from a peripheral blood sample.Methods for detecting single B cells that are producing an antibody withthe desired specificity are well known in the art, for example, byplaque formation, fluorescence-activated cell sorting, in vitrostimulation followed by detection of specific antibody, and the like.Methods for selection of specific antibody producing B cells include,for example, preparing a single cell suspension of B cells in soft agarthat contains SOST or a peptide fragment thereof. Binding of thespecific antibody produced by the B cell to the antigen results in theformation of a complex, which may be visible as an immunoprecipitate.After the B cells producing the specific antibody are selected, thespecific antibody genes may be cloned by isolating and amplifying DNA ormRNA according to methods known in the art and described herein.

For particular uses, fragments of anti-TGF-beta binding proteinantibodies may be desired. Antibody fragments, F(ab′)₂ Fab, Fab′, Fv,Fc, Fd, retain the antigen binding site of the whole antibody andtherefore bind to the same epitope. These antigen-binding fragmentsderived from an antibody can be obtained, for example, by proteolytichydrolysis of the antibody, for example, pepsin or papain digestion ofwhole antibodies according to conventional methods. As an illustration,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent to produce 3.5S Fab′monovalent fragments. Optionally, the cleavage reaction can be performedusing a blocking group for the sulfhydryl groups that result fromcleavage of disulfide linkages. As an alternative, an enzymatic cleavageusing papain produces two monovalent Fab fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods inEnzymology 1:422 (Academic Press 1967); and by Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4. Other methods for cleaving antibodies,such as separating heavy chains to form monovalent light-heavy chainfragments (Fd), further cleaving of fragments, or other enzymatic,chemical, or genetic techniques may also be used, so long as thefragments bind to the antigen that is recognized by the intact antibody.

An antibody fragment may also be any synthetic or genetically engineeredprotein that acts like an antibody in that it binds to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (scFvproteins), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. The antibody of thepresent invention preferably comprises at least one variable regiondomain. The variable region domain may be of any size or amino acidcomposition and will generally comprise at least one hypervariable aminoacid sequence responsible for antigen binding and which is adjacent toor in frame with one or more framework sequences. In general terms, thevariable (V) region domain may be any suitable arrangement ofimmunoglobulin heavy (V_(H)) and/or light (V_(L)) chain variabledomains. Thus, for example, the V region domain may be monomeric and bea V_(H) or V_(L) domain, which is capable of independently bindingantigen with acceptable affinity. Alternatively, the V region domain maybe dimeric and contain V_(H)-V_(H), V_(H) V_(L), or V_(L)-V_(L), dimers.Preferably, the V region dimer comprises at least one V_(H) and at leastone V_(L) chain that are non-covalently associated (hereinafter referredto as F_(v)). If desired, the chains may be covalently coupled eitherdirectly, for example via a disulphide bond between the two variabledomains, or through a linker, for example a peptide linker, to form asingle chain Fv (scF_(v)).

The variable region domain may be any naturally occurring variabledomain or an engineered version thereof. By engineered version is meanta variable region domain that has been created using recombinant DNAengineering techniques. Such engineered versions include those created,for example, from a specific antibody variable region by insertions,deletions, or changes in or to the amino acid sequences of the specificantibody. Particular examples include engineered variable region domainscontaining at least one CDR and optionally one or more framework aminoacids from a first antibody and the remainder of the variable regiondomain from a second antibody.

The variable region domain may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example, a V_(H) domain that is present in the variable regiondomain may be linked to an immunoglobulin C_(H)1 domain, or a fragmentthereof. Similarly a V_(L) domain may be linked to a C_(K) domain or afragment thereof. In this way, for example, the antibody may be a Fabfragment wherein the antigen binding domain contains associated V_(H)and V_(L) domains covalently linked at their C-termini to a CH1 andC_(K) domain, respectively. The CH1 domain may be extended with furtheramino acids, for example to provide a hinge region or a portion of ahinge region domain as found in a Fab′ fragment, or to provide furtherdomains, such as antibody CH2 and CH3 domains.

Another form of an antibody fragment is a peptide comprising for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing polynucleotides thatencode the CDR of an antibody of interest. Such polynucleotides areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region using mRNA of antibody-producing cells asa template (see, for example, Larrick et al., Methods: A Companion toMethods in Enzymology 2:106, 1991; Courtenay-Luck, “Genetic Manipulationof Monoclonal Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), page 166(Cambridge University Press 1995); and Ward et al., “GeneticManipulation and Expression of Antibodies,” in Monoclonal Antibodies:Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss,Inc. 1995)).

Alternatively, the antibody may be a recombinant or engineered antibodyobtained by the use of recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Such DNA is known and/or is readily available from DNAlibraries including for example phage-antibody libraries (see Chiswelland McCafferty, Tibtech. 10:80-84 (1992)) or if desired can besynthesized. Standard molecular biology and/or chemistry procedures maybe used to sequence and manipulate the DNA, for example, to introducecodons to create cysteine residues, or to modify, add or delete otheramino acids or domains as desired.

Chimeric antibodies, specific for a TGF-beta binding protein, and whichinclude humanized antibodies, may also be generated according to thepresent invention. A chimeric antibody has at least one constant regiondomain derived from a first mammalian species and at least one variableregion domain derived from a second, distinct mammalian species (see,e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-55 (1984)).In preferred embodiments, a chimeric antibody may be constructed bycloning the polynucleotide sequence that encodes at least one variableregion domain derived from a non-human monoclonal antibody, such as thevariable region derived from a murine, rat, or hamster monoclonalantibody, into a vector containing a nucleotide sequence that encodes atleast one human constant region (see, e.g., Shin et al., MethodsEnzymol. 178:459-76 (1989); Walls et al., Nucleic Acids Res. 21:2921-29(1993)). By way of example, the polynucleotide sequence encoding thelight chain variable region of a murine monoclonal antibody may beinserted into a vector containing a nucleotide sequence encoding thehuman kappa light chain constant region sequence. In a separate vector,the polynucleotide sequence encoding the heavy chain variable region ofthe monoclonal antibody may be cloned in frame with sequences encoding ahuman IgG constant region, for example, the human IgG1 constant region.The particular human constant region selected may depend upon theeffector functions desired for the particular antibody (e.g., complementfixing, binding to a particular Fc receptor, etc.). Preferably, theconstructed vectors will be transfected into eukaryotic cells for stableexpression of the chimeric antibody. Another method known in the art forgenerating chimeric antibodies is homologous recombination (e.g., U.S.Pat. No. 5,482,856).

A non-human/human chimeric antibody may be further geneticallyengineered to create a “humanized” antibody. Such a humanized antibodymay comprise a plurality of CDRs derived from an immunoglobulin of anon-human mammalian species, at least one human variable frameworkregion, and at least one human immunoglobulin constant region. Usefulstrategies for designing humanized antibodies may include, for exampleby way of illustration and not limitation, identification of humanvariable framework regions that are most homologous to the non-humanframework regions of the chimeric antibody. Without wishing to be boundby theory, such a strategy may increase the likelihood that thehumanized antibody will retain specific binding affinity for a TGF-betabinding protein, which in some preferred embodiments may besubstantially the same affinity for a TGF-beta binding protein orvariant or fragment thereof, and in certain other preferred embodimentsmay be a greater affinity for TGF-beta binding protein. See, e.g., Joneset al., 1986 Nature 321:522-25; Riechmann et al., 1988 Nature332:323-27. Designing such a humanized antibody may therefore includedetermining CDR loop conformations and structural determinants of thenon-human variable regions, for example, by computer modeling, and thencomparing the CDR loops and determinants to known human CDR loopstructures and determinants. See, e.g., Padian et al., 1995 FASEB9:133-39; Chothia et al., 1989 Nature, 342:377-383. Computer modelingmay also be used to compare human structural templates selected bysequence homology with the non-human variable regions. See, e.g.,Bajorath et al., 1995 Ther. Immunol. 2:95-103; EP-0578515-A3. Ifhumanization of the non-human CDRs results in a decrease in bindingaffinity, computer modeling may aid in identifying specific amino acidresidues that could be changed by site-directed or other mutagenesistechniques to partially, completely or supra-optimally (i.e., increaseto a level greater than that of the non-humanized antibody) restoreaffinity. Those having ordinary skill in the art are familiar with thesetechniques, and will readily appreciate numerous variations andmodifications to such design strategies.

One such method for preparing a humanized antibody is called veneering.As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site thatretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.,Ann. Rev. Biochem. 59:439-73, 1990. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residuesthat are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. Initially, the FRs of thevariable domains of an antibody to molecule of interest are comparedwith corresponding FR sequences of human variable domains obtained fromthe above-identified sources. The most homologous human V regions arethen compared residue by residue to corresponding murine amino acids.The residues in the murine FR that differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues that mayhave a significant effect on the tertiary structure of V region domains,such as proline, glycine, and charged amino acids.

In this manner, the resultant “veneered” antigen-binding sites are thusdesigned to retain the rodent CDR residues, the residues substantiallyadjacent to the CDRs, the residues identified as buried or mostly buried(solvent inaccessible), the residues believed to participate innon-covalent (e.g., electrostatic and hydrophobic) contacts betweenheavy and light chain domains, and the residues from conservedstructural regions of the FRs which are believed to influence the“canonical” tertiary structures of the CDR loops. These design criteriaare then used to prepare recombinant nucleotide sequences that combinethe CDRs of both the heavy and light chain of a antigen-binding siteinto human-appearing FRs that can be used to transfect mammalian cellsfor the expression of recombinant human antibodies that exhibit theantigen specificity of the rodent antibody molecule.

An additional method for selecting antibodies that specifically bind toa TGF-beta binding protein or variant or fragment thereof is by phagedisplay. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55;Burton et al., 1994 Adv. Immunol. 57:191-280. Human or murineimmunoglobulin variable region gene combinatorial libraries may becreated in phage vectors that can be screened to select Ig fragments(Fab, Fv, sFv, or multimers thereof) that bind specifically to TGF-betabinding protein or variant or fragment thereof. See, e.g., U.S. Pat. No.5,223,409; William D. Huse et al., “Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda,” Science246:1275-1281, December 1989; see also L. Sastry et al., “Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc. Natl. Acad. Sci. USA 86:5728-5732,August 1989; see also Michelle Alting-Mees et al., “Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas,” Strategies inMolecular Biology 3:1-9, January 1990; Kang et al., 1991 Proc. Natl.Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol.227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and referencescited therein). A commercial system is available from Stratagene (LaJolla, Calif.) which enables the production of antibodies throughrecombinant techniques. Briefly, mRNA is isolated from a B cellpopulation, and utilized to create heavy and light chain immunoglobulincDNA expression libraries in the λ ImmunoZap(H) and λImmunoZap(L)vectors. Positive plaques may subsequently be converted to a non-lyticplasmid which allows high level expression of monoclonal antibodyfragments from E. coli. Alternatively, a library containing a pluralityof polynucleotide sequences encoding Ig variable region fragments may beinserted into the genome of a filamentous bacteriophage, such as M13 ora variant thereof, in frame with the sequence encoding a phage coatprotein. A fusion protein may be a fusion of the coat protein with thelight chain variable region domain and/or with the heavy chain variableregion domain. According to certain embodiments, immunoglobulin Fabfragments may also be displayed on a phage particle (see, e.g., U.S.Pat. No. 5,698,426). These vectors may be screened individually orco-expressed to form Fab fragments or antibodies (see Huse et al.,supra; see also Sastry et al., supra).

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to incorporate the variableregions of a gene which encodes a specifically binding antibody. Withinone embodiment, the genes which encode the variable region from ahybridoma producing a monoclonal antibody of interest are amplifiedusing nucleotide primers for the variable region. These primers may besynthesized by one of ordinary skill in the art, or may be purchasedfrom commercially available sources. Stratagene (La Jolla, Calif.) sellsprimers for mouse and human variable regions including, among others,primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd), C_(H1), V_(L) and C_(L)regions. These primers may be utilized to amplify heavy or light chainvariable regions, which may then be inserted into vectors such asImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectorsmay then be introduced into E. coli, yeast, or mammalian-based systemsfor expression. Utilizing these techniques, large amounts of asingle-chain protein containing a fusion of the V_(H) and V_(L) domainsmay be produced (see Bird et al., Science 242:423-426, 1988). Inaddition, such techniques may be utilized to change a “murine” antibodyto a “human” antibody, without altering the binding specificity of theantibody.

In certain particular embodiments of the invention, combinatorial phagelibraries may also be used for humanization of non-human variableregions. See, e.g., Rosok et al., 1996 J. Biol. Chem. 271:22611-18;Rader et al., 1998 Proc. Natl. Acad. Sci. USA 95:8910-15. A phagelibrary may be screened to select an Ig variable region fragment ofinterest by immunodetection methods known in the art and describedherein, and the DNA sequence of the inserted immunoglobulin gene in thephage so selected may be determined by standard techniques. See,Sambrook et al., 2001 Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press. The selected Ig-encoding sequence may then becloned into another suitable vector for expression of the Ig fragmentor, optionally, may be cloned into a vector containing Ig constantregions, for expression of whole immunoglobulin chains.

In certain other embodiments, the invention contemplates SOST-specificantibodies that are multimeric antibody fragments. Useful methodologiesare described generally, for example in Hayden et al. 1997, Curr Opin.Immunol. 9:201-12; Coloma et al., 1997 Nat. Biotechnol. 15:159-63). Forexample, multimeric antibody fragments may be created by phagetechniques to form miniantibodies (U.S. Pat. No. 5,910,573) or diabodies(Holliger et al., 1997, Cancer Immunol. Immunother. 45:128-130).

In certain embodiments of the invention, an antibody specific for SOSTmay be an antibody that is expressed as an intracellular protein. Suchintracellular antibodies are also referred to as intrabodies and maycomprise an Fab fragment, or preferably comprise a scFv fragment (see,e.g., Lecerf et al., Proc. Natl. Acad. Sci. USA 98:4764-49 (2001). Theframework regions flanking the CDR regions can be modified to improveexpression levels and solubility of an intrabody in an intracellularreducing environment (see, e.g., Worn et al., J. Biol. Chem.275:2795-803 (2000). An intrabody may be directed to a particularcellular location or organelle, for example by constructing a vectorthat comprises a polynucleotide sequence encoding the variable regionsof an intrabody that may be operatively fused to a polynucleotidesequence that encodes a particular target antigen within the cell (see,e.g., Graus-Porta et al., Mol. Cell. Biol. 15:1182-91 (1995); Lener etal., Eur. J. Biochem. 267:1196-205 (2000)). An intrabody may beintroduced into a cell by a variety of techniques available to theskilled artisan including via a gene therapy vector, or a lipid mixture(e.g., Provectin™ manufactured by Imgenex Corporation, San Diego,Calif.), or according to photochemical internalization methods.

Introducing amino acid mutations into an immunoglobulin moleculespecific for a TGF-beta binding protein may be useful to increase thespecificity or affinity for TGF-beta binding protein or to alter aneffector function. Immunoglobulins with higher affinity for TGF-betabinding protein may be generated by site-directed mutagenesis ofparticular residues. Computer assisted three-dimensional molecularmodeling may be employed to identify the amino acid residues to bechanged, in order to improve affinity for the TGF-beta binding protein.See, e.g., Mountain et al., 1992, Biotechnol. Genet. Eng. Rev. 10:1-142.Alternatively, combinatorial libraries of CDRs may be generated in M13phage and screened for immunoglobulin fragments with improved affinity.See, e.g., Glaser et al., 1992, J. Immunol. 149:3903-3913; Barbas etal., 1994 Proc. Natl. Acad. Sci. USA 91:3809-13; U.S. Pat. No.5,792,456.

Effector functions may also be altered by site-directed mutagenesis.See, e.g., Duncan et al., 1988 Nature 332:563-64; Morgan et al., 1995Immunology 86:319-24; Eghtedarzedeh-Kondri et al., 1997 Biotechniques23:830-34. For example, mutation of the glycosylation site on the Fcportion of the immunoglobulin may alter the ability of theimmunoglobulin to fix complement. See, e.g., Wright et al., 1997 TrendsBiotechnol. 15:26-32. Other mutations in the constant region domains mayalter the ability of the immunoglobulin to fix complement, or to effectantibody-dependent cellular cytotoxicity. See, e.g., Duncan et al., 1988Nature 332:563-64; Morgan et al., 1995 Immunology 86:319-24; Sensel etal., 1997 Mol. Immunol. 34:1019-29.

According to certain embodiments, non-human, human, or humanized heavychain and light chain variable regions of any of the Ig moleculesdescribed herein may be constructed as single chain Fv (scFv)polypeptide fragments (single chain antibodies). See, e.g., Bird et al.,1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883. Multi-functional scFv fusion proteins may be generated bylinking a polynucleotide sequence encoding an scFv polypeptide in-framewith at least one polynucleotide sequence encoding any of a variety ofknown effector proteins. These methods are known in the art, and aredisclosed, for example, in EP-B1-0318554, U.S. Pat. No. 5,132,405, U.S.Pat. No. 5,091,513, and U.S. Pat. No. 5,476,786. By way of example,effector proteins may include immunoglobulin constant region sequences.See, e.g., Hollenbaugh et al., 1995 J. Immunol. Methods 188:1-7. Otherexamples of effector proteins are enzymes. As a non-limiting example,such an enzyme may provide a biological activity for therapeuticpurposes (see, e.g., Siemers et al., 1997 Bioconjug. Chem. 8:510-19), ormay provide a detectable activity, such as horseradishperoxidase-catalyzed conversion of any of a number of well-knownsubstrates into a detectable product, for diagnostic uses. Still otherexamples of scFv fusion proteins include Ig-toxin fusions, orimmunotoxins, wherein the scFv polypeptide is linked to a toxin.

The scFv or any antibody fragment described herein may, in certainembodiments, be fused to peptide or polypeptide domains that permitsdetection of specific binding between the fusion protein and antigen(e.g., a TGF-beta binding protein). For example, the fusion polypeptidedomain may be an affinity tag polypeptide for detecting binding of thescFv fusion protein to a TGF-beta binding protein by any of a variety oftechniques with which those skilled in the art will be familiar.Examples of a peptide tag, include avidin, streptavidin or His (e.g.,polyhistidine). Detection techniques may also include, for example,binding of an avidin or streptavidin fusion protein to biotin or to abiotin mimetic sequence (see, e.g., Luo et al., 1998 J. Biotechnol.65:225 and references cited therein), direct covalent modification of afusion protein with a detectable moiety (e.g., a labeling moiety),non-covalent binding of the fusion protein to a specific labeledreporter molecule, enzymatic modification of a detectable substrate by afusion protein that includes a portion having enzyme activity, orimmobilization (covalent or non-covalent) of the fusion protein on asolid-phase support. Other useful affinity polypeptides for constructionof scFv fusion proteins may include streptavidin fusion proteins, asdisclosed, for example, in WO 89/03422, U.S. Pat. No. 5,489,528, U.S.Pat. No. 5,672,691, WO 93/24631, U.S. Pat. No. 5,168,049, U.S. Pat. No.5,272,254; avidin fusion proteins (see, e.g., EP 511,747); an enzymesuch as glutathione-S-transferase; and Staphylococcus aureus protein Apolypeptide.

The polynucleotides encoding an antibody or fragment thereof thatspecifically bind a TGF-beta binding protein, as described herein, maybe propagated and expressed according to any of a variety of well-knownprocedures for nucleic acid excision, ligation, transformation, andtransfection using any number of known expression vectors. Thus, incertain embodiments expression of an antibody fragment may be preferredin a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun etal., 1989 Methods Enzymol. 178:497-515). In certain other embodiments,expression of the antibody or a fragment thereof may be preferred in aeukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Pichia pastoris), animal cells (includingmammalian cells) or plant cells. Examples of suitable animal cellsinclude, but are not limited to, myeloma (such as a mouse NSO line),COS, CHO, or hybridoma cells. Examples of plant cells include tobacco,corn, soybean, and rice cells.

Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), ColdSpring Harbor Laboratory Press, 1988). Suitable techniques includepeptide or protein affinity columns (including use of anti-constantregion antibodies attached to the column matrix), HPLC or RP-HPLC,purification on protein A or protein G columns, or any combination ofthese techniques.

c. Mutant TGF-Beta Binding-Proteins

As described herein and below in the Examples (e.g., Examples 8 and 9),altered versions of TGF-beta binding-protein which compete with nativeTGF-beta binding-protein's ability to block the activity of a particularTGF-beta family member should lead to increased bone density. Thus,mutants of TGF-beta binding-protein which bind to the TGF-beta familymember but do not inhibit the function of the TGF-beta family memberwould meet the criteria. The mutant versions must effectively competewith the endogenous inhibitory functions of TGF-beta binding-protein.

d. Production of Proteins

Polypeptides described herein include the TGF binding protein sclerostinand variants thereof and antibodies or fragments thereof thatspecifically bind to sclerostin. The polynucleotides that encode thesepolypeptides include derivatives of the genes that are substantiallysimilar to the genes and isolated nucleic acid molecules, and, whenappropriate, the proteins (including peptides and polypeptides) that areencoded by the genes and their derivatives. As used herein, a nucleotidesequence is deemed to be “substantially similar” if (a) the nucleotidesequence is derived from the coding region of the above-described genesand nucleic acid molecules and includes, for example, portions of thesequence or allelic variations of the sequences discussed above, oralternatively, encodes a molecule which inhibits the binding of TGF-betabinding-protein to a member of the TGF-beta family; (b) the nucleotidesequence is capable of hybridization to nucleotide sequences of thepresent invention under moderate, high or very high stringency (seeSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, NY, 1989); and/or (c) the DNA sequencesare degenerate as a result of the genetic code to the DNA sequencesdefined in (a) or (b). Further, the nucleic acid molecule disclosedherein includes both complementary and non-complementary sequences,provided the sequences otherwise meet the criteria set forth herein.Within the context of the present invention, high stringency meansstandard hybridization conditions (e.g., 5×SSPE, 0.5% SDS at 65° C., orthe equivalent). The structure of the proteins encoded by the nucleicacid molecules described herein may be predicted from the primarytranslation products using the hydrophobicity plot function of, forexample, P/C Gene or Intelligenetics Suite (Intelligenetics, MountainView, Calif.), or according to the methods described by Kyte andDoolittle (J. Mol. Biol. 157:105-132, 1982).

Proteins of the present invention may be prepared in the form of acidicor basic salts, or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Moreover, due to degeneracy in the genetic code, for example,there may be considerable variation in nucleotide sequences encoding thesame amino acid sequence.

Other derivatives of the proteins disclosed herein include conjugates ofthe proteins along with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins which may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al., Bio/Technology 6:1204, 1988.) Alternatively, fusion proteinssuch as Flag®/TGF-beta binding-protein be constructed in order to assistin the identification, expression, and analysis of the protein.

Proteins of the present invention may be constructed using a widevariety of techniques described herein. Further, mutations may beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene or nucleic acid molecule having particular codons altered accordingto the substitution, deletion, or insertion required. Exemplary methodsof making the alterations set forth above are disclosed by Walder et al.(Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik(BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering:Principles and Methods, Plenum Press, 1981); and Sambrook et al.(supra). Deletion or truncation derivatives of proteins (e.g., a solubleextracellular portion) may also be constructed by utilizing convenientrestriction endonuclease sites adjacent to the desired deletion.Subsequent to restriction, overhangs may be filled in and the DNAreligated. Exemplary methods of making the alterations set forth aboveare disclosed by Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989).

Mutations which are made in the nucleic acid molecules of the presentinvention preferably preserve the reading frame of the coding sequences.Furthermore, the mutations will preferably not create complementaryregions that when transcribed could hybridize to produce secondary mRNAstructures, such as loops or hairpins, that would adversely affecttranslation of the mRNA. Although a mutation site may be predetermined,it is not necessary that the nature of the mutation per se bepredetermined. For example, in order to select for optimumcharacteristics of mutants at a given site, random mutagenesis may beconducted at the target codon and the expressed mutants screened forgain or loss or retention of biological activity. Alternatively,mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes a derivativehaving the desired amino acid insertion, substitution, or deletion.

Nucleic acid molecules which encode proteins of the present inventionmay also be constructed utilizing techniques such as PCR mutagenesis,chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402-3406,1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene88:107-111, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., Genome 3:112-117, 1989).

The present invention also provides for the manipulation and expressionof the above described genes and nucleic acid molecules by culturinghost cells containing a vector capable of expressing the above-describedgenes. Such vectors or vector constructs include either synthetic orcDNA-derived nucleic acid molecules encoding the desired protein, whichare operably linked to suitable transcriptional or translationalregulatory elements. Suitable regulatory elements may be derived from avariety of sources, including bacterial, fungal, viral, mammalian,insect, or plant genes. Selection of appropriate regulatory elements isdependent on the host cell chosen, and may be readily accomplished byone of ordinary skill in the art. Examples of regulatory elementsinclude a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a transcriptional terminator, and a ribosomal bindingsequence, including a translation initiation signal.

Nucleic acid molecules that encode any of the proteins described abovemay be readily expressed by a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,insect, or plant cells. Methods for transforming or transfecting suchcells to express foreign DNA are well known in the art (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., Proc. Natl.Acad. Sci. USA 75:1929-1933, 1978; Murray et al., U.S. Pat. No.4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S.Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel etal., U.S. Pat. No. 4,766,075; and Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989;for plant cells see Czako and Marton, Plant Physiol. 104:1067-1071,1994; and Paszkowski et al., Biotech. 24:387-392, 1992).

Bacterial host cells suitable for carrying out the present inventioninclude E. coli, B. subtilis, Salmonella typhimurium, and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,as well as many other bacterial species well known to one of ordinaryskill in the art and described herein. A representative example of abacterial host cell includes E. coli DH5α (Stratagene, LaJolla, Calif.).

Bacterial expression vectors preferably comprise a promoter whichfunctions in the host cell, one or more selectable phenotypic markers,and a bacterial origin of replication. Representative promoters includethe β-lactamase (penicillinase) and lactose promoter system (see Changet al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studieret al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin etal., Gene 87:123-126, 1990), the tip promoter (Nichols and Yanofsky,Meth. in Enzymology 101:155, 1983), and the tac promoter (Russell etal., Gene 20:231, 1982). Representative selectable markers includevarious antibiotic resistance markers such as the kanamycin orampicillin resistance genes. Many plasmids suitable for transforminghost cells are well known in the art, including among others, pBR322(see Bolivar et al., Gene 2:95, 1977), the pUC plasmids pUC18, pUC19,pUC118, pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983 andVieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a,and Bluescript M13 (Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others, Saccharomyces pombe, Saccharomycescerevisiae, the genera Pichia or Kluyveromyces and various species ofthe genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349).Suitable expression vectors for yeast and fungi include, among others,YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl.Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene8:121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978)and derivatives thereof.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum,New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples ofuseful promoters for fungi vectors include those derived fromAspergillus nidulans glycolytic genes, such as the adh3 promoter(McKnight et al., EMBO J. 4:2093-2099, 1985). The expression units mayalso include a transcriptional terminator. An example of a suitableterminator is the adh3 terminator (McKnight et al., supra, 1985).

As with bacterial vectors, the yeast vectors will generally include aselectable marker, which may be one of any number of genes that exhibita dominant phenotype for which a phenotypic assay exists to enabletransformants to be selected. Preferred selectable markers are thosethat complement host cell auxotrophy, provide antibiotic resistance, orenable a cell to utilize specific carbon sources, and include leu2(Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3(Struhl et al., ibid.). Another suitable selectable marker is the catgene, which confers chloramphenicol resistance on yeast cells.

Techniques for transforming fungi are well known in the literature andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc.Natl. Acad. Sci. USA 81:1740-1747, 1984), and Russell (Nature301:167-169, 1983). The genotype of the host cell may contain a geneticdefect that is complemented by the selectable marker present on theexpression vector. Choice of a particular host and selectable marker iswell within the level of ordinary skill in the art.

Protocols for the transformation of yeast are also well known to thoseof ordinary skill in the art. For example, transformation may be readilyaccomplished either by preparation of spheroplasts of yeast with DNA(see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment withalkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163,1983). Transformation of fungi may also be carried out usingpolyethylene glycol as described by Cullen et al. (Bio/Technology 5:369,1987).

Viral vectors include those that comprise a promoter that directs theexpression of an isolated nucleic acid molecule that encodes a desiredprotein as described above. A wide variety of promoters may be utilizedwithin the context of the present invention, including for example,promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviralpromoter (Ohno et al., Science 265:781-784, 1994), neomycinphosphotransferase promoter/enhancer, late parvovirus promoter (Koeringet al., Hum. Gene Therap. 5:457-463, 1994), Herpes TK promoter, SV40promoter, metallothionein IIa gene enhancer/promoter, cytomegalovirusimmediate early promoter, and the cytomegalovirus immediate latepromoter. Within particularly preferred embodiments of the invention,the promoter is a tissue-specific promoter (see e.g., WO 91/02805; EP0,415,731; and WO 90/07936). Representative examples of suitable tissuespecific promoters include neural specific enolase promoter, plateletderived growth factor beta promoter, bone morphogenic protein promoter,human alpha1-chimaerin promoter, synapsin I promoter and synapsin IIpromoter. In addition to the above-noted promoters, other viral-specificpromoters (e.g., retroviral promoters (including those noted above, aswell as others such as HIV promoters), hepatitis, herpes (e.g., EBV),and bacterial, fungal or parasitic (e.g., malarial)-specific promotersmay be utilized in order to target a specific cell or tissue which isinfected with a virus, bacteria, fungus, or parasite.

Mammalian cells suitable for carrying out the present invention include,among others COS, CHO, SaOS, osteosarcomas, KS483, MG-63, primaryosteoblasts, and human or mammalian bone marrow stroma. Mammalianexpression vectors for use in carrying out the present invention willinclude a promoter capable of directing the transcription of a clonedgene, nucleic acid molecule, or cDNA. Preferred promoters include viralpromoters and cellular promoters. Bone specific promoters include thepromoter for bone sialo-protein and the promoter for osteocalcin. Viralpromoters include the cytomegalovirus immediate early promoter (Boshartet al., Cell 41:521-530, 1985), cytomegalovirus immediate late promoter,SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), MMTVLTR, RSV LTR, metallothionein-1, adenovirus E1a. Cellular promotersinclude the mouse metallothionein-1 promoter (Palmiter et al., U.S. Pat.No. 4,579,821), a mouse V_(K) promoter (Bergman et al., Proc. Natl.Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nucleic Acids Res.15:5496, 1987) and a mouse V_(H) promoter (Loh et al., Cell 33:85-93,1983). The choice of promoter will depend, at least in part, upon thelevel of expression desired or the recipient cell line to betransfected.

Such expression vectors may also contain a set of RNA splice siteslocated downstream from the promoter and upstream from the DNA sequenceencoding the peptide or protein of interest. Preferred RNA splice sitesmay be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the coding sequence of interest. Suitable polyadenylationsignals include the early or late polyadenylation signals from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theAdenovirus 5 E1B region and the human growth hormone gene terminator(DeNoto et al., Nucleic Acids Res. 9:3719-3730, 1981). The expressionvectors may include a noncoding viral leader sequence, such as theAdenovirus 2 tripartite leader, located between the promoter and the RNAsplice sites. Preferred vectors may also include enhancer sequences,such as the SV40 enhancer. Expression vectors may also include sequencesencoding the adenovirus VA RNAs. Suitable expression vectors can beobtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

Vector constructs comprising cloned DNA sequences can be introduced intocultured mammalian cells by, for example, calcium phosphate-mediatedtransfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson,Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845,1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,1987). To identify cells that have stably transfected with the vector orhave integrated the cloned DNA, a selectable marker is generallyintroduced into the cells along with the gene or cDNA of interest.Preferred selectable markers for use in cultured mammalian cells includegenes that confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. Preferred amplifiable selectable markers are the DHFR gene andthe neomycin resistance gene. Selectable markers are reviewed by Thilly(Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.,which is incorporated herein by reference).

Mammalian cells containing a suitable vector are allowed to grow for aperiod of time, typically 1-2 days, to begin expressing the DNAsequence(s) of interest. Drug selection is then applied to select forgrowth of cells that are expressing the selectable marker in a stablefashion. For cells that have been transfected with an amplifiable,selectable marker, the drug concentration may be increased in a stepwisemanner to select for increased copy number of the cloned sequences,thereby increasing expression levels. Cells expressing the introducedsequences are selected and screened for production of the protein ofinterest in the desired form or at the desired level. Cells that satisfythese criteria can then be cloned and scaled up for production.

Protocols for the transfection of mammalian cells are well known tothose of ordinary skill in the art. Representative methods includecalcium phosphate mediated transfection, electroporation, lipofection,retroviral, adenoviral and protoplast fusion-mediated transfection (seeSambrook et al., supra). Naked vector constructs can also be taken up bymuscular cells or other suitable cells subsequent to injection into themuscle of a mammal (or other animals). Methods for using insect hostcells and plant host cells for production of polypeptides are known inthe art and described herein. Numerous insect host cells known in theart can also be useful within the present invention. For example, theuse of baculoviruses as vectors for expressing heterologous DNAsequences in insect cells has been reviewed by Atkinson et al. (Pestic.Sci. 28:215-224, 1990). Numerous vectors and plant host cells known inthe art can also be useful within the present invention, for example,the use of Agrobacterium rhizogenes as vectors for expressing genes andnucleic acid molecules in plant cells (see review by Sinkar et al., J.Biosci. (Bangalore 11:47-58, 1987).

Within related aspects of the present invention, proteins of the presentinvention may be expressed in a transgenic animal whose germ cells andsomatic cells contain a gene which encodes the desired protein and whichis operably linked to a promoter effective for the expression of thegene. Alternatively, in a similar manner transgenic animals may beprepared that lack the desired gene (e.g., “knock-out” mice). Suchtransgenics may be prepared in a variety of non-human animals, includingmice, rats, rabbits, sheep, dogs, goats, and pigs (see Hammer et al.,Nature 315:680-683, 1985, Palmiter et al., Science 222:809-814, 1983,Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985, Palmiterand Brinster, Cell 41:343-345, 1985, and U.S. Pat. Nos. 5,175,383,5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384).Briefly, an expression vector, including a nucleic acid molecule to beexpressed together with appropriately positioned expression controlsequences, is introduced into pronuclei of fertilized eggs, for example,by microinjection. Integration of the injected DNA is detected by blotanalysis of DNA from tissue samples. It is preferred that the introducedDNA be incorporated into the germ line of the animal so that it ispassed on to the animal's progeny. Tissue-specific expression may beachieved through the use of a tissue-specific promoter, or through theuse of an inducible promoter, such as the metallothionein gene promoter(Palmiter et al., 1983, supra), which allows regulated expression of thetransgene.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsas described herein. Supernatants from such cell lines, or proteininclusions, or whole cells from which the protein is not excreted intothe supernatant, can then be treated by a variety of purificationprocedures in order to isolate the desired proteins. For example, thesupernatant may be first concentrated using commercially availableprotein concentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as, for example, ananti-protein antibody (e.g., an antibody that specifically binds to thepolypeptide to be isolated) bound to a suitable support. Alternatively,anion or cation exchange resins may be employed in order to purify theprotein. As a further alternative, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps may be employed tofurther purify the protein. Other methods of isolating the proteins ofthe present invention are well known in the art.

The purity of an isolated polypeptide may be determined by techniquesknown in the art and described herein, such as gel electrophoresis andchromatography methods. Preferably, such isolated polypeptides are atleast about 90% pure, more preferably at least about 95% pure, and mostpreferably at least about 99% pure. Within certain specific embodiments,a protein is deemed to be “isolated” within the context of the presentinvention if no other undesired protein is detected pursuant to SDS-PAGEanalysis followed by Coomassie blue staining. Within other embodiments,the desired protein can be isolated such that no other undesired proteinis detected pursuant to SDS-PAGE analysis followed by silver staining.

3. Nucleic Acid Molecules

Within other aspects of the invention, nucleic acid molecules areprovided which are capable of inhibiting TGF-beta binding-proteinbinding to a member of the TGF-beta family. For example, within oneembodiment antisense oligonucleotide molecules are provided whichspecifically inhibit expression of TGF-beta binding-protein nucleic acidsequences (see generally, Hirashima et al. in Molecular Biology of RNA:New Perspectives (M. Inouye and B. S. Dudock, eds., 1987 Academic Press,San Diego, p. 401); Oligonucleotides: Antisense Inhibitors of GeneExpression (J. S. Cohen, ed., 1989 MacMillan Press, London); Stein andCheng, Science 261:1004-1012, 1993; WO 95/10607; U.S. Pat. No.5,359,051; WO 92/06693; and EP-A2-612844). Briefly, such molecules areconstructed such that they are complementary to, and able to formWatson-Crick base pairs with, a region of transcribed TGF-betabinding-protein mRNA sequence. The resultant double-stranded nucleicacid interferes with subsequent processing of the mRNA, therebypreventing protein synthesis (see Example 10).

Within other aspects of the invention, ribozymes are provided which arecapable of inhibiting the TGF-beta binding-protein binding to a memberof the TGF-beta family. As used herein, “ribozymes” are intended toinclude RNA molecules that contain anti-sense sequences for specificrecognition, and an RNA-cleaving enzymatic activity. The catalyticstrand cleaves a specific site in a target RNA at greater thanstoichiometric concentration. A wide variety of ribozymes may beutilized within the context of the present invention, including forexample, the hammerhead ribozyme (for example, as described by Forsterand Symons, Cell 48:211-220, 1987; Haseloff and Gerlach, Nature328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988; Haseloffand Gerlach, Nature 334:585, 1988); the hairpin ribozyme (for example,as described by Haseloff et al., U.S. Pat. No. 5,254,678, issued Oct.19, 1993 and Hempel et al., European Patent Publication No. 0 360 257,published Mar. 26, 1990); and Tetrahymena ribosomal RNA-based ribozymes(see Cech et al., U.S. Pat. No. 4,987,071). Ribozymes of the presentinvention typically consist of RNA, but may also be composed of DNA,nucleic acid analogs (e.g., phosphorothioates), or chimerics thereof(e.g., DNA/RNA/RNA).

4. Labels

The gene product or any of the candidate molecules described above andbelow, may be labeled with a variety of compounds, including forexample, fluorescent molecules, toxins, and radionuclides.Representative examples of fluorescent molecules include fluorescein,Phycobili proteins, such as phycoerythrin, rhodamine, Texas red andluciferase. Representative examples of toxins include ricin, abrindiphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein,tritin, Shigella toxin, and Pseudomonas exotoxin A. Representativeexamples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97,Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188,Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. In addition, theantibodies described above may also be labeled or conjugated to onepartner of a ligand binding pair. Representative examples includeavidin-biotin, streptavidin-biotin, and riboflavin-riboflavin bindingprotein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981; Antibody Conjugate, U.S. Pat. No. 5,106,951;Fluorogenic Materials and Labeling Techniques, U.S. Pat. No. 4,018,884;Metal Radionuclide Labeled Proteins for Diagnosis and Therapy, U.S. Pat.No. 4,897,255; and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics, U.S. Pat. No. 4,988,496; see also Inman, Methods InEnzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B,Jakoby and Wilchek (eds.), Academic Press, New York, p. 30, 1974; seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1-32, 1988).

Pharmaceutical Compositions

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules which inhibits the TGF-beta binding-protein binding to amember of the TGF-beta family along with a pharmaceutically orphysiologically acceptable carrier, excipients or diluents. Generally,such carriers should be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, maltose, sucrose or dextrins, chelating agents suchas EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

The pharmaceutical compositions of the present invention may be preparedfor administration by a variety of different routes. In general, thetype of carrier is selected based on the mode of administration.Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, oral, nasal,intrathecal, rectal, vaginal, sublingual or parenteral administration,including subcutaneous, intravenous, intramuscular, intrasternal,intracavernous, intrameatal, or intraurethral injection or infusion. Apharmaceutical composition (e.g., for oral administration or delivery byinjection) may be in the form of a liquid (e.g., an elixir, syrup,solution, emulsion or suspension). A liquid pharmaceutical compositionmay include, for example, one or more of the following: sterile diluentssuch as water for injection, saline solution, preferably physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils that mayserve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents;antioxidants; chelating agents; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. The use of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile.

The compositions described herein may be formulated for sustainedrelease (i.e., a formulation such as a capsule or sponge that effects aslow release of compound following administration). Such compositionsmay generally be prepared using well known technology and administeredby, for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Sustained-release formulationsmay contain an agent dispersed in a carrier matrix and/or containedwithin a reservoir surrounded by a rate controlling membrane. Carriersfor use within such formulations are biocompatible, and may also bebiodegradable; preferably the formulation provides a relatively constantlevel of active component release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented. Illustrativecarriers useful in this regard include microparticles ofpoly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose,dextran and the like. Other illustrative delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638).

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems, such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers or as controlled release matrices for thecompositions of this invention. Exemplary calcium phosphate particlesare disclosed, for example, in published patent application No.WO/0046147.

For pharmaceutical compositions comprising a polynucleotide encoding ananti-SOST antibody and/or modulating agent (such that the polypeptideand/or modulating agent is generated in situ), the polynucleotide may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid, and bacterial, viraland mammalian expression systems. Techniques for incorporating DNA intosuch expression systems are well known to those of ordinary skill in theart. The DNA may also be “naked,” as described, for example, in Ulmer etal., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences, 15th ed., pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. Moreover, for human administration, preparations will ofcourse preferably meet sterility, pyrogenicity, and the general safetyand purity standards as required by FDA Office of Biologics standards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol. 16(7):307-21, 1998;Takakura, Nippon Rinsho 56(3):691-95, 1998; Chandran et al., Indian J.Exp. Biol. 35(8):801-09, 1997; Margalit, Crit. Rev. Ther. Drug CarrierSyst. 12(2-3):233-61, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No.5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J. Biol. Chem. 265(27):16337-42, 1990; Muller et al., DNA CellBiol. 9(3):221-29, 1990). In addition, liposomes are free of the DNAlength constraints that are typical of viral-based delivery systems.Liposomes have been used effectively to introduce genes, various drugs,radiotherapeutic agents, enzymes, viruses, transcription factors,allosteric effectors and the like, into a variety of cultured cell linesand animals. Furthermore, the use of liposomes does not appear to beassociated with autoimmune responses or unacceptable toxicity aftersystemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev. Ind. Pharm. 24(12):1113-28, 1998). To avoid side effectsdue to intracellular polymeric overloading, such ultrafine particles(sized around 0.1 μm) may be designed using polymers able to be degradedin vivo. Such particles can be made as described, for example, byCouvreur et al., Crit. Rev. Ther. Drug Carrier Syst. 5(1):1-20, 1988;zur Muhlen et al., Eur. J. Pharm. Biophamm. 45(2):149-55, 1998; Zambauxet al., J. Controlled Release 50(1-3):31-40, 1998; and U.S. Pat. No.5,145,684.

In addition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material that providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute thepharmaceutical composition.

Methods of Treatment

The present invention also provides methods for increasing the mineralcontent and mineral density of bone. Briefly, numerous conditions resultin the loss of bone mineral content, including for example, disease,genetic predisposition, accidents which result in the lack of use ofbone (e.g., due to fracture), therapeutics which effect bone resorption,or which kill bone forming cells and normal aging. Through use of themolecules described herein which inhibit the TGF-beta binding-proteinbinding to a TGF-beta family member such conditions may be treated orprevented. As utilized herein, it should be understood that bone mineralcontent has been increased if bone mineral content has been increased ina statistically significant manner (e.g., greater than one-half standarddeviation), at a selected site.

A wide variety of conditions that result in loss of bone mineral contentmay be treated with the molecules described herein. Patients with suchconditions may be identified through clinical diagnosis utilizing wellknown techniques (see, e.g., Harrison's Principles of Internal Medicine,McGraw-Hill, Inc.). Representative examples of diseases that may betreated included dysplasias, wherein there is abnormal growth ordevelopment of bone. Representative examples of such conditions includeachondroplasia, cleidocranial dysostosis, enchondromatosis, fibrousdysplasia, Gaucher's Disease, hypophosphatemic rickets, Marfan'sSyndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesisimperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions,fractures, periodontal disease, pseudoarthrosis, and pyogenicosteomyelitis.

Other conditions which may be treated or prevented include a widevariety of causes of osteopenia (i.e., a condition that causes greaterthan one standard deviation of bone mineral content or density belowpeak skeletal mineral content at youth). Representative examples of suchconditions include anemic states, conditions caused by steroids,conditions caused by heparin, bone marrow disorders, scurvy,malnutrition, calcium deficiency, idiopathic osteoporosis, congenitalosteopenia or osteoporosis, alcoholism, chronic liver disease, senility,postmenopausal state, oligomenorrhea, amenorrhea, pregnancy, diabetesmellitus, hyperthyroidism, Cushing's disease, acromegaly, hypogonadism,immobilization or disuse, reflex sympathetic dystrophy syndrome,transient regional osteoporosis, and osteomalacia.

Within one aspect of the present invention, bone mineral content ordensity may be increased by administering to a warm-blooded animal atherapeutically effective amount of a molecule that inhibits binding ofthe TGF-beta binding-protein to a TGF-beta family member. Examples ofwarm-blooded animals that may be treated include both vertebrates andmammals, including for example humans, horses, cows, pigs, sheep, dogs,cats, rats and mice. Representative examples of therapeutic moleculesinclude ribozymes, ribozyme genes, antisense oligonucleotides, andantibodies (e.g., humanized antibodies or any other antibody describedherein).

Within other aspects of the present invention, methods are provided forincreasing bone density, comprising the steps of introducing into cellswhich home to bone, a vector that directs the expression of a moleculewhich inhibits binding of the TGF-beta binding-protein to a member ofthe TGF-beta family, and administering the vector-containing cells to awarm-blooded animal. Briefly, cells that home to bone may be obtaineddirectly from the bone of patients (e.g., cells obtained from the bonemarrow such as CD34+, osteoblasts, osteocytes, and the like), fromperipheral blood, or from cultures.

A vector that directs the expression of a molecule that inhibits thebinding of TGF-beta binding-protein to a member of the TGF-beta familymay be introduced into cells. Representative examples of suitablevectors include viral vectors such as herpes viral vectors (e.g., U.S.Pat. No. 5,288,641), adenoviral vectors (e.g., WO 94/26914, WO 93/9191;Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993;Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993;Caillaud et al., Eur. J. Neurosci. 5(10:1287-1291, 1993; Vincent et al.,Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378,1992; and Levrero et al., Gene 101(2):195-202, 1991), adeno-associatedviral vectors (WO 95/13365; Flotte et al., PNAS 90(22):10613-10617,1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum.Gene Therap. 5:457-463, 1994), pox virus vectors (Panicali and Paoletti,PNAS 79:4927-4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm.193(2):653-660, 1993), and retroviruses (e.g., EP 0,415,731; WO90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat.No. 5,219,740; WO 93/11230; WO 93/10218). Viral vectors may likewise beconstructed which contain a mixture of different elements (e.g.,promoters, envelope sequences, and the like) from different viruses, ornon-viral sources. Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below. Within otherembodiments of the invention, nucleic acid molecules which encode amolecule which inhibits binding of the TGF-beta binding-protein to amember of the TGF-beta family may be administered by a variety oftechniques, including, for example, administration of asialoosomucoid(ASOR) conjugated with poly-L-lysine DNA complexes (Cristano et al.,PNAS 92122-92126, 1993), DNA linked to killed adenovirus (Curiel et al.,Hum. Gene Ther. 3(2):147-154, 1992), cytofectin-mediated introduction(DMRIE-DOPE, Vical, Calif.), direct DNA injection (Acsadi et al., Nature352:815-818, 1991); DNA ligand (Wu et al., J. of Biol. Chem.264:16985-16987, 1989); lipofection (Feigner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417, 1989); liposomes (Pickering et al., Circ.89(1):13-21, 1994; and Wang et al., PNAS 84:7851-7855, 1987);microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991);and direct delivery of nucleic acids which encode the protein itselfeither alone (Vile and Hart, Cancer Res. 53: 3860-3864, 1993), orutilizing PEG-nucleic acid complexes. Representative examples ofmolecules that may be expressed by the vectors of present inventioninclude ribozymes and antisense molecules, each of which are discussedin more detail above.

Determination of increased bone mineral content may be determineddirectly through the use of X-rays (e.g., Dual Energy X-rayAbsorptometry or “DEXA”), or by inference through bone turnover markers(such as osteoblast specific alkaline phosphatase, osteocalcin, type 1procollagen, C′ propeptide (PICP), and total alkaline phosphatase; seeComier, C., Curr. Opin. in Rheu. 7:243, 1995), or by markers of boneresorption (pyridinoline, deoxypryridinoline, N-telopeptide, urinaryhydroxyproline, plasma tartrate-resistant acid phosphatases andgalactosyl hydroxylysine; see Comier, supra). The amount of bone massmay also be calculated from body weights or by other methods known inthe art (see Guinness-Hey, Metab. Bone Dis. and Relat. Res. 5:177-181,1984).

As will be evident to one of skill in the art, the amount and frequencyof administration will depend, of course, on such factors as the natureand severity of the indication being treated, the desired response, thecondition of the patient, and so forth. Typically, the compositions maybe administered by a variety of techniques, as noted above.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Sclerosteosis Maps to the Long Arm of HumanChromosome 17

Genetic mapping of the defect responsible for sclerosteosis in humanslocalized the gene responsible for this disorder to the region of humanchromosome 17 that encodes a novel TGF-beta binding-protein familymember. In sclerosteosis, skeletal bone displays a substantial increasein mineral density relative to that of unafflicted individuals. Bone inthe head displays overgrowth as well. Sclerosteosis patients aregenerally healthy although they may exhibit variable degrees ofsyndactyly at birth and variable degrees of cranial compression andnerve compression in the skull.

Linkage analysis of the gene defect associated with sclerosteosis wasconducted by applying the homozygosity mapping method to DNA samplescollected from 24 South African Afrikaaner families in which the diseaseoccurred. (Sheffield et al., 1994, Human Molecular Genetics 3:1331-1335.“Identification of a Bardet-Biedl syndrome locus on chromosome 3 andevaluation of an efficient approach to homozygosity mapping”). TheAfrikaaner population of South Africa is genetically homogeneous; thepopulation is descended from a small number of founders who colonizedthe area several centuries ago, and it has been isolated by geographicand social barriers since the founding. Sclerosteosis is rare everywherein the world outside the Afrikaaner community, which suggests that amutation in the gene was present in the founding population and hassince increased in numbers along with the increase in the population.The use of homozygosity mapping is based on the assumption that DNAmapping markers adjacent to a recessive mutation are likely to behomozygous in affected individuals from consanguineous families andisolated populations.

A set of 371 microsatellite markers (Research Genetics, Set 6) from theautosomal chromosomes was selected to type pools of DNA fromsclerosteosis patient samples. The DNA samples for this analysis camefrom 29 sclerosteosis patients in 24 families, 59 unaffected familymembers and a set of unrelated control individuals from the samepopulation. The pools consisted of 4-6 individuals, either affectedindividuals, affected individuals from consanguineous families, parentsand unaffected siblings, or unrelated controls. In the pools ofunrelated individuals and in most of the pools with affected individualsor family members analysis of the markers showed several allele sizesfor each marker. One marker, D17S1299, showed an indication ofhomozygosity: one band in several of the pools of affected individuals.

All 24 sclerosteosis families were typed with a total of 19 markers inthe region of D17S1299 (at 17q12-q21). Affected individuals from everyfamily were shown to be homozygous in this region, and 25 of the 29individuals were homozygous for a core haplotype; they each had the samealleles between D17S1787 and D17S930. The other four individuals had onechromosome which matched this haplotype and a second which did not. Insum, the data compellingly suggested that this 3 megabase regioncontained the sclerosteosis mutation. Sequence analysis of most of theexons in this 3 megabase region identified a nonsense mutation in thenovel TGF-beta binding-protein coding sequence (C>T mutation at position117 of Sequence ID No. 1 results in a stop codon). This mutation wasshown to be unique to sclerosteosis patients and carriers of Afrikaanerdescent. The identity of the gene was further confirmed by identifying amutation in its intron (A>T mutation at position +3 of the intron) whichresults in improper mRNA processing in a single, unrelated patient withdiagnosed sclerosteosis.

Example 2 Tissue-Specificity of TGF-Beta Binding-Protein Gene ExpressionA. Human Beer Gene Expression by RT-PCR:

First-strand cDNA was prepared from the following total RNA samplesusing a commercially available kit (“Superscript Preamplification Systemfor First-Strand cDNA Synthesis”, Life Technologies, Rockville, Md.):human brain, human liver, human spleen, human thymus, human placenta,human skeletal muscle, human thyroid, human pituitary, human osteoblast(NHOst from Clonetics Corp., San Diego, Calif.), human osteosarcoma cellline (Saos-2, ATCC# HTB-85), human bone, human bone marrow, humancartilage, vervet monkey bone, saccharomyces cerevisiae, and humanperipheral blood monocytes. All RNA samples were purchased from acommercial source (Clontech, Palo Alto, Calif.), except the followingwhich were prepared in-house: human osteoblast, human osteosarcoma cellline, human bone, human cartilage and vervet monkey bone. These in-houseRNA samples were prepared using a commercially available kit (“TRIReagent”, Molecular Research Center, Inc., Cincinnati, Ohio).

PCR was performed on these samples, and additionally on a human genomicsample as a control. The sense Beer oligonucleotide primer had thesequence 5′-CCGGAGCTGGAGAACAACAAG-3′ (SEQ ID NO:19). The antisense Beeroligonucleotide primer had the sequence 5′-GCACTGGCCGGAGCACACC-3′ (SEQID NO:20). In addition, PCR was performed using primers for the humanbeta-actin gene, as a control. The sense beta-actin oligonucleotideprimer had the sequence 5′-AGGCCAACCGCGAGAAGATGA CC-3′ (SEQ ID NO:21).The antisense beta-actin oligonucleotide primer had the sequence5′-GAAGT CCAGGGCGACGTAGCA-3′ (SEQ ID NO:22). PCR was performed usingstandard conditions in 25 ul reactions, with an annealing temperature of61 degrees Celsius. Thirty-two cycles of PCR were performed with theBeer primers and twenty-four cycles were performed with the beta-actinprimers.

Following amplification, 12 ul from each reaction were analyzed byagarose gel electrophoresis and ethidium bromide staining. See FIG. 2A.

B. RNA In-Situ Hybridization of Mouse Embryo Sections:

The full length mouse Beer cDNA (Sequence ID No. 11) was cloned into thepCR2.1 vector (Invitrogen, Carlsbad, Calif.) in the antisense and sensedirection using the manufacturer's protocol. ³⁵S-alpha-GTP-labeled cRNAsense and antisense transcripts were synthesized using in-vitrotranscription reagents supplied by Ambion, Inc (Austin, Tex.). In-situhybridization was performed according to the protocols of Lyons et al.(J. Cell Biol. 111:2427-2436, 1990).

The mouse Beer cRNA probe detected a specific message expressed in theneural tube, limb buds, blood vessels and ossifying cartilages ofdeveloping mouse embryos. Panel A in FIG. 3 shows expression in theapical ectodermal ridge (aer) of the limb (1) bud, blood vessels (by)and the neural tube (nt). Panel B shows expression in the 4^(th)ventricle of the brain (4). Panel C shows expression in the mandible(ma) cervical vertebrae (cv), occipital bone (oc), palate (pa) and ablood vessel (bv). Panel D shows expression in the ribs (r) and a heartvalve (va). Panel A is a transverse section of 10.5 dpc embryo. Panel Bis a sagittal section of 12.5 dpc embryo and panels C and D are sagittalsections of 15.5 dpc embryos.

ba=branchial arch, h=heart, te=telencephalon (forebrain), b=brain,f=frontonasal mass, g=gut, h=heart, j=jaw, li=liver, lu=lung, ot=oticvesicle, ao=, sc=spinal cord, skm=skeletal muscle, ns=nasal sinus,th=thymus, to=tongue, fl=forelimb, di=diaphragm

Example 3 Expression and Purification of Recombinant Beer Protein A.Expression in COS-1 Cells:

The DNA sequence encoding the full length human Beer protein wasamplified using the following PCR oligonucleotide primers: The 5′oligonucleotide primer had the sequence 5′-AAGCTTGGTACCATGCAGCTCCCAC-3′(SEQ ID NO:23) and contained a HindIII restriction enzyme site (in bold)followed by 19 nucleotides of the Beer gene starting 6 base pairs priorto the presumed amino terminal start codon (ATG). The 3′ oligonucleotideprimer had the sequence 5′-AAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCGTAGGCGTTCTCCAGCT-3′ (SEQ ID NO:24) and contained a HindIIIrestriction enzyme site (in bold) followed by a reverse complement stopcodon (CTA) followed by the reverse complement of the FLAG epitope(underlined, Sigma-Aldrich Co., St. Louis, Mo.) flanked by the reversecomplement of nucleotides coding for the carboxy terminal 5 amino acidsof the Beer. The PCR product was TA cloned (“Original TA Cloning Kit”,Invitrogen, Carlsbad, Calif.) and individual clones were screened by DNAsequencing. A sequence-verified clone was then digested by HindIII andpurified on a 1.5% agarose gel using a commercially available reagents(“QIAquick Gel Extraction Kit”, Qiagen Inc., Valencia, Calif.). Thisfragment was then ligated to HindIII digested, phosphatase-treatedpcDNA3.1 (Invitrogen, Carlsbad, Calif.) plasmid with T4 DNA ligase.DH10B E. coli were transformed and plated on LB, 100 μg/ml ampicillinplates. Colonies bearing the desired recombinant in the properorientation were identified by a PCR-based screen, using a 5′ primercorresponding to the T7 promoter/priming site in pcDNA3.1 and a 3′primer with the sequence 5′-GCACTGGCCGGAGCACACC-3′ (SEQ ID NO:25) thatcorresponds to the reverse complement of internal BEER sequence. Thesequence of the cloned fragment was confirmed by DNA sequencing.

COS-1 cells (ATCC# CRL-1650) were used for transfection. 50 μg of theexpression plasmid pcDNA-Beer-Flag was transfected using a commerciallyavailable kit following protocols supplied by the manufacturer(“DEAE-Dextran Transfection Kit”, Sigma Chemical Co., St. Louis, Mo.).The final media following transfection was DMEM (Life Technologies,Rockville, Md.) containing 0.1% Fetal Bovine Serum. After 4 days inculture, the media was removed. Expression of recombinant BEER wasanalyzed by SDS-PAGE and Western Blot using anti-FLAG® M2 monoclonalantibody (Sigma-Aldrich Co., St. Louis, Mo.). Purification ofrecombinant BEER protein was performed using an anti-FLAG M2 affinitycolumn (“Mammalian Transient Expression System”, Sigma-Aldrich Co., St.Louis, Mo.). The column profile was analyzed via SDS-PAGE and WesternBlot using anti-FLAG M2 monoclonal antibody.

B. Expression in SF9 Insect Cells:

The human Beer gene sequence was amplified using PCR with standardconditions and the following primers:

Sense primer: (SEQ ID NO: 26)5′-GTCGTCGGATCCATGGGGTGGCAGGCGTTCAAGAATGAT-3′ Antisense primer: (SEQ IDNO: 27) 5′-GTCGTCAAGCTTCTACTTGTCATCGTCCTTGTAGTCGTAGGCGTTCT CCAGCTCGGC-3′

The resulting cDNA contained the coding region of Beer with twomodifications. The N-terminal secretion signal was removed and a FLAGepitope tag (Sigma) was fused in frame to the C-terminal end of theinsert. BamH 1 and HindIII cloning sites were added and the gene wassubcloned into pMelBac vector (Invitrogen) for transfer into abaculoviral expression vector using standard methods.

Recombinant baculoviruses expressing Beer protein were made using theBac-N-Blue transfection kit (Invitrogen) and purified according to themanufacturers instructions.

SF9 cells (Invitrogen) were maintained in TNM_FH media (Invitrogen)containing 10% fetal calf serum. For protein expression, SF9 cultures inspinner flasks were infected at an MOI of greater than 10. Samples ofthe media and cells were taken daily for five days, and Beer expressionmonitored by western blot using an anti-FLAG M2 monoclonal antibody(Sigma) or an anti-Beer rabbit polyclonal antiserum.

After five days the baculovirus-infected SF9 cells were harvested bycentrifugation and cell associated protein was extracted from the cellpellet using a high salt extraction buffer (1.5 M NaCl, 50 mM Tris pH7.5). The extract (20 ml per 300 ml culture) was clarified bycentrifugation, dialyzed three times against four liters of Trisbuffered saline (150 mM NaCl, 50 mM Tris pH 7.5), and clarified bycentrifugation again. This high salt fraction was applied to HitrapHeparin (Pharmacia; 5 ml bed volume), washed extensively with HEPESbuffered saline (25 mM HEPES 7.5, 150 mM Nacl) and bound proteins wereeluted with a gradient from 150 mM NaCl to 1200 mM NaCl. Beer elutionwas observed at aproximately 800 mM NaCl. Beer containing fractions weresupplemented to 10% glycerol and 1 mM DTT and frozen at −80 degrees C.

Example 4 Preparation and Testing of Polyclonal Antibodies to Beer,Gremlin, and Dan A. Preparation of Antigen:

The DNA sequences of Human Beer, Human Gremlin, and Human Dan wereamplified using standard PCR methods with the following oligonucleotideprimers:

H. Beer Sense: (SEQ ID NO: 28) 5′-GACTTGGATCCCAGGGGTGGCAGGCGTTC-3′Antisense (SEQ ID NO: 29) 5′-AGCATAAGCTTCTAGTAGGCGTTCTCCAG-3′ H. GremlinSense: (SEQ ID NO: 30) 5′-GACTTGGATCCGAAGGGAAAAAGAAAGGG-3′ Antisense:(SEQ ID NO: 31) 5′-AGCATAAGCTTTTAATCCAAATCGATGGA-3′ H. Dan Sense: (SEQID NO: 32) 5′-ACTACGAGCTCGGCCCCACCACCCATCAACAAG-3′ Antisense: (SEQ IDNO: 33) 5′-ACTTAGAAGCTTTCAGTCCTCAGCCCCCTCTTCC-3′

In each case the listed primers amplified the entire coding region minusthe secretion signal sequence. These include restriction sites forsubcloning into the bacterial expression vector pQE-30 (Qiagen Inc.,Valencia, Calif.) at sites BamHI/HindIII for Beer and Gremlin, and sitesSacI/HindIII for Dan. pQE30 contains a coding sequence for a 6×His tagat the 5′ end of the cloning region. The completed constructs weretransformed into E. coli strain M-15/pRep (Qiagen Inc) and individualclones verified by sequencing. Protein expression in M-15/pRep andpurification (6×His affinity tag binding to Ni-NTA coupled to Sepharose)were performed as described by the manufacturer (Qiagen, TheQIAexpressionist).

The E. coli-derived Beer protein was recovered in significant quantityusing solubilization in 6M guanidine and dialyzed to 2-4M to preventprecipitation during storage. Gremlin and Dan protein were recovered inhigher quantity with solubilization in 6M guanidine and a postpurification guanidine concentration of 0.5M.

B. Production and Testing of Polyclonal Antibodies:

Polyclonal antibodies to each of the three antigens were produced inrabbit and in chicken hosts using standard protocols (R & R Antibody,Stanwood, Wash.; standard protocol for rabbit immunization and antiserarecovery; Short Protocols in Molecular Biology. 2nd edition. 1992.11.37-11.41. Contributors Helen M. Cooper and Yvonne Paterson; chickenantisera was generated with Strategic Biosolutions, Ramona, Calif.).

Rabbit antisera and chicken egg Igy fraction were screened for activityvia Western blot. Each of the three antigens was separated by PAGE andtransferred to 0.45 um nitrocellulose (Novex, San Diego, Calif.). Themembrane was cut into strips with each strip containing approximately 75ng of antigen. The strips were blocked in 3% Blotting Grade Block(Bio-Rad Laboratories, Hercules, Calif.) and washed 3 times in 1×Trisbuffer saline (TBS)/0.02% TWEEN buffer. The primary antibody(preimmunization bleeds, rabbit antisera or chicken egg IgY in dilutionsranging from 1:100 to 1:10,000 in blocking buffer) was incubated withthe strips for one hour with gentle rocking. A second series of threewashes 1×TBS/0.02% TWEEN was followed by an one hour incubation with thesecondary antibody (peroxidase conjugated donkey anti-rabbit, AmershamLife Science, Piscataway, N.J.; or peroxidase conjugated donkeyanti-chicken, Jackson ImmunoResearch, West Grove, Pa.). A final cycle of3× washes of 1×TBS/0.02% TWEEN was performed and the strips weredeveloped with Lumi-Light Western Blotting Substrate (Roche MolecularBiochemicals, Mannheim, Germany).

C. Antibody Cross-Reactivity Test:

Following the protocol described in the previous section, nitrocellulosestrips of Beer, Gremlin or Dan were incubated with dilutions (1:5000 and1:10,000) of their respective rabbit antisera or chicken egg IgY as wellas to antisera or chicken egg Igy (dilutions 1:1000 and 1:5000) made tothe remaining two antigens. The increased levels of nonmatchingantibodies was performed to detect low affinity binding by thoseantibodies that may be seen only at increased concentration. Theprotocol and duration of development is the same for all three bindingevents using the protocol described above. There was no antigencross-reactivity observed for any of the antigens tested.

Example 5 Interaction of Beer with TGF-Beta Super-Family Proteins

The interaction of Beer with proteins from different phylogenetic armsof the TGF-β superfamily were studied using immunoprecipitation methods.Purified TGFβ-1, TGFβ-2, TGFβ-3, BMP-4, BMP-5, BMP-6 and GDNF wereobtained from commerical sources (R&D systems; Minneapolis, Minn.). Arepresentative protocol is as follows. Partially purified Beer wasdialyzed into HEPES buffered saline (25 mM HEPES 7.5, 150 mM NaCl).Immunoprecipitations were done in 300 ul of IP buffer (150 mM NaCl, 25mM Tris pH 7.5, 1 mM EDTA, 1.4 mM β-mercaptoethanol, 0.5% triton×100,and 10% glycerol). 30 ng recombinant human BMP-5 protein (R&D systems)was applied to 15 ul of FLAG affinity matrix (Sigma; St Louis Mo.)) inthe presence and absence of 500 ng FLAG epitope-tagged Beer. Theproteins were incubated for 4 hours @ 4° C. and then the affinitymatrix-associated proteins were washed 5 times in IP buffer (1 ml perwash). The bound proteins were eluted from the affinity matrix in 60microliters of 1×SDS PAGE sample buffer. The proteins were resolved bySDS PAGE and Beer associated BMP-5 was detected by western blot usinganti-BMP-5 antiserum (Research Diagnostics, Inc) (see FIG. 5).

Beer Ligand Binding Assay:

FLAG-Beer protein (20 ng) is added to 100 ul PBS/0.2% BSA and adsorbedinto each well of 96 well microtiter plate previously coated withanti-FLAG monoclonal antibody (Sigma; St Louis Mo.) and blocked with 10%BSA in PBS. This is conducted at room temperature for 60 minutes. Thisprotein solution is removed and the wells are washed to remove unboundprotein. BMP-5 is added to each well in concentrations ranging from 10μM to 500 nM in PBS/0.2% BSA and incubated for 2 hours at roomtemperature. The binding solution is removed and the plate washed withthree times with 200 ul volumes of PBS/0.2% BSA. BMP-5 levels are thendetected using BMP-5 anti-serum via ELISA (F. M. Ausubel et al (1998)Current Protocols in Mol. Biol. Vol 2 11.2.1-11.2.22). Specific bindingis calculated by subtracting non-specific binding from total binding andanalyzed by the LIGAND program (Munson and Podbard, Anal. Biochem., 107,p 220-239, (1980).

In a variation of this method, Beer is engineered and expressed as ahuman Fc fusion protein. Likewise the ligand BMP is engineered andexpressed as mouse Fc fusion. These proteins are incubated together andthe assay conducted as described by Mellor et al using homogeneous timeresolved fluorescence detection (G. W. Mellor et al., J of BiomolScreening, 3(2) 91-99, 1998).

Example 6 Screening Assay for Inhibition of TGF-Beta Binding-ProteinBinding to TGF-Beta Family Members

The assay described above is replicated with two exceptions. First, BMPconcentration is held fixed at the Kd determined previously. Second, acollection of antagonist candidates is added at a fixed concentration(20 uM in the case of the small organic molecule collections and 1 uM inantibody studies). These candidate molecules (antagonists) of TGF-betabinding-protein binding include organic compounds derived fromcommercial or internal collections representing diverse chemicalstructures. These compounds are prepared as stock solutions in DMSO andare added to assay wells at ≦1% of final volume under the standard assayconditions. These are incubated for 2 hours at room temperature with theBMP and Beer, the solution removed and the bound BMP is quantitated asdescribed. Agents that inhibit 40% of the BMP binding observed in theabsence of compound or antibody are considered antagonists of thisinteraction. These are further evaluated as potential inhibitors basedon titration studies to determine their inhibition constants and theirinfluence on TGF-beta binding-protein binding affinity. Comparablespecificity control assays may also be conducted to establish theselectivity profile for the identified antagonist through studies usingassays dependent on the BMP ligand action (e.g. BMP/BMP receptorcompetition study).

Example 7 Inhibition of TGF-Beta Binding-Protein Localization to BoneMatrix

Evaluation of inhibition of localization to bone matrix (hydroxyapatite)is conducted using modifications to the method of Nicolas (Nicolas, V.Calif Tissue Int 57:206, 1995). Briefly, ¹²⁵I-labelled TGF-betabinding-protein is prepared as described by Nicolas (supra).Hydroxyapatite is added to each well of a 96 well microtiter plateequipped with a polypropylene filtration membrane (Polyfiltroninc,Weymouth Mass.). TGF-beta binding-protein is added to 0.2% albumin inPBS buffer. The wells containing matrix are washed 3 times with thisbuffer. Adsorbed TGF-beta binding-protein is eluted using 0.3M NaOH andquantitated.

Inhibitor identification is conducted via incubation of TGF-betabinding-protein with test molecules and applying the mixture to thematrix as described above. The matrix is washed 3 times with 0.2%albumin in PBS buffer. Adsorbed TGF-beta binding-protein is eluted using0.3 M NaOH and quantitated. Agents that inhibit 40% of the TGF-betabinding-protein binding observed in the absence of compound or antibodyare considered bone localization inhibitors. These inhibitors arefurther characterized through dose response studies to determine theirinhibition constants and their influence on TGF-beta binding-proteinbinding affinity.

Example 8 Construction of TGF-Beta Binding-Protein Mutant A.Mutagenesis:

A full-length TGF-beta binding-protein cDNA in pBluescript SK serves asa template for mutagenesis. Briefly, appropriate primers (see thediscussion provided above) are utilized to generate the DNA fragment bypolymerase chain reaction using Vent DNA polymerase (New EnglandBiolabs, Beverly, Mass.). The polymerase chain reaction is run for 23cycles in buffers provided by the manufacturer using a 57° C. annealingtemperature. The product is then exposed to two restriction enzymes andafter isolation using agarose gel electrophoresis, ligated back intopRBP4-503 from which the matching sequence has been removed by enzymaticdigestion. Integrity of the mutant is verified by DNA sequencing.

B. Mammalian Cell Expression and Isolation of Mutant TGF-BetaBinding-Protein:

The mutant TGF-beta binding-protein cDNAs are transferred into thepcDNA3.1 mammalian expression vector described in EXAMPLE 3. Afterverifying the sequence, the resultant constructs are transfected intoCOS-1 cells, and secreted protein is purified as described in EXAMPLE 3.

Example 9 Animal Models—I Generation of Transgenic Mice Overexpressingthe Beer Gene

The ˜200 kilobase (kb) BAC clone 15G5, isolated from the CITB mousegenomic DNA library (distributed by Research Genetics, Huntsville, Ala.)was used to determine the complete sequence of the mouse Beer gene andits 5′ and 3′ flanking regions. A 41 kb SalI fragment, containing theentire gene body, plus ˜17 kb of 5′ flanking and ˜20 kb of 3′ flankingsequence was sub-cloned into the BamHI site of the SuperCosI cosmidvector (Stratagene, La Jolla, Calif.) and propagated in the E. colistrain DH10B. From this cosmid construct, a 35 kb MluI-AviII restrictionfragment (Sequence No. 6), including the entire mouse Beer gene, as wellas 17 kb and 14 kb of 5′ and 3′ flanking sequence, respectively, wasthen gel purified, using conventional means, and used for microinjectionof mouse zygotes (DNX Transgenics; U.S. Pat. No. 4,873,191). Founderanimals in which the cloned DNA fragment was integrated randomly intothe genome were obtained at a frequency of 5-30% of live-born pups. Thepresence of the transgene was ascertained by performing Southern blotanalysis of genomic DNA extracted from a small amount of mouse tissue,such as the tip of a tail. DNA was extracted using the followingprotocol: tissue was digested overnight at 55° C. in a lysis buffercontaining 200 mM NaCl, 100 mM Tris pH8.5, 5 mM EDTA, 0.2% SDS and 0.5mg/ml Proteinase K. The following day, the DNA was extracted once withphenol/chloroform (50:50), once with chloroform/isoamylalcohol (24:1)and precipitated with ethanol. Upon resuspension in TE (10 mM TrispH7.5, 1 mM EDTA) 8-10 ug of each DNA sample were digested with arestriction endonuclease, such as EcoRI, subjected to gelelectrophoresis and transferred to a charged nylon membrane, such asHyBondN+ (Amersham, Arlington Heights, Ill.). The resulting filter wasthen hybridized with a radioactively labelled fragment of DNA derivingfrom the mouse Beer gene locus, and able to recognize both a fragmentfrom the endogenous gene locus and a fragment of a different sizederiving from the transgene. Founder animals were bred to normalnon-transgenic mice to generate sufficient numbers of transgenic andnon-transgenic progeny in which to determine the effects of Beer geneoverexpression. For these studies, animals at various ages (for example,1 day, 3 weeks, 6 weeks, 4 months) are subjected to a number ofdifferent assays designed to ascertain gross skeletal formation, bonemineral density, bone mineral content, osteoclast and osteoblastactivity, extent of endochondral ossification, cartilage formation, etc.The transcriptional activity from the transgene may be determined byextracting RNA from various tissues, and using an RT-PCR assay whichtakes advantage of single nucleotide polymorphisms between the mousestrain from which the transgene is derived (129 Sv/J) and the strain ofmice used for DNA microinjection [(C57BL5/J×SJL/J)F2].

Animal Models—II Disruption of the Mouse Beer Gene by HomologousRecombination

Homologous recombination in embryonic stem (ES) cells can be used toinactivate the endogenous mouse Beer gene and subsequently generateanimals carrying the loss-of-function mutation. A reporter gene, such asthe E. coli β-galactosidase gene, was engineered into the targetingvector so that its expression is controlled by the endogenous Beergene's promoter and translational initiation signal. In this way, thespatial and temporal patterns of Beer gene expression can be determinedin animals carrying a targeted allele.

The targeting vector was constructed by first cloning thedrug-selectable phosphoglycerate kinase (PGK) promoter drivenneomycin-resistance gene (neo) cassette from pGT-N29 (New EnglandBiolabs, Beverly, Mass.) into the cloning vector pSP72 (Promega, Madson,Wis.). PCR was used to flank the PGKneo cassette with bacteriophage P1loxP sites, which are recognition sites for the P1 Cre recombinase(Hoess et al., PNAS USA, 79:3398, 1982). This allows subsequent removalof the neo-resistance marker in targeted ES cells or ES cell-derivedanimals (U.S. Pat. No. 4,959,317). The PCR primers were comprised of the34 nucleotide (ntd) loxP sequence, 15-25 ntd complementary to the 5′ and3′ ends of the PGKneo cassette, as well as restriction enzymerecognition sites (BamHI in the sense primer and EcoRI in the anti-senseprimer) for cloning into pSP72. The sequence of the sense primer was5′-AATCTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATCTGCAGGATTCGAGGGCCCCT-3′(SEQ ID NO:34); sequence of the anti-sense primer was5′-AATCTGAATTCCACCGGTGTTAATTAAATAACTTCGTATAATGTATGCTATACGAAGTTATAGATCTAGAGTCAGCTTCTGA-3′ (SEQ ID NO:35).

The next step was to clone a 3.6 kb XhoI-HindIII fragment, containingthe E. coli β-galactosidase gene and SV40 polyadenylation signal frompSVβ (Clontech, Palo Alto, Calif.) into the pSP72-PGKneo plasmid. The“short arm” of homology from the mouse Beer gene locus was generated byamplifying a 2.4 kb fragment from the BAC clone 15G5. The 3′ end of thefragment coincided with the translational initiation site of the Beergene, and the anti-sense primer used in the PCR also included 30 ntdcomplementary to the 5′ end of the β-galactosidase gene so that itscoding region could be fused to the Beer initiation site in-frame. Theapproach taken for introducing the “short arm” into thepSP72-βgal-PGKneo plasmid was to linearize the plasmid at a siteupstream of the β-gal gene and then to co-transform this fragment withthe “short arm” PCR product and to select for plasmids in which the PCRproduct was integrated by homologous recombination. The sense primer forthe “short arm” amplification included 30 ntd complementary to the pSP72vector to allow for this recombination event. The sequence of the senseprimer was5′-ATTTAGGTGACACTATAGAACTCGAGCAGCTGAAGCTTAACCACATGGTGGCTCACAACCAT-3′(SEQ ID NO:36) and the sequence of the anti-sense primer was5′-AACGACGGCCAGTGAATCCGTAATCATGGTCATGCTGCCAGGTGGAGGAGGGCA-3′ (SEQ IDNO:37).

The “long arm” from the Beer gene locus was generated by amplifying a6.1 kb fragment from BAC clone 15G5 with primers which also introducethe rare-cutting restriction enzyme sites SgrAI, FseI, AscI and PacI.Specifically, the sequence of the sense primer was5′-ATTACCACCGGTGACACCCGCTTCCTGACAG-3′ (SEQ ID NO:38); the sequence ofthe anti-sense primer was5′-ATTACTTAATTAAACATGGCGCGCCATATGGCCGGCCCCTAATTGCGGCGCATCGTTAATT-3′ (SEQID NO:39). The resulting PCR product was cloned into the TA vector(Invitrogen, Carlsbad, Calif.) as an intermediate step.

The mouse Beer gene targeting construct also included a secondselectable marker, the herpes simplex virus I thymidine kinase gene(HSVTK) under the control of rous sarcoma virus long terminal repeatelement (RSV LTR). Expression of this gene renders mammalian cellssensitive (and inviable) to gancyclovir; it is therefore a convenientway to select against neomycin-resistant cells in which the constructhas integrated by a non-homologous event (U.S. Pat. No. 5,464,764). TheRSVLTR-HSVTK cassette was amplified from pPS1337 using primers thatallow subsequent cloning into the FseI and AscI sites of the “longarm”-TA vector plasmid. For this PCR, the sequence of the sense primerwas 5′-ATTACGGCCGGCCGCAAAGGAATTCAAGATCTGA-3′ (SEQ ID NO:40); thesequence of the anti-sense primer was5′-ATTACGGCGCGCCCCTCACAGGCCGCACCCAGCT-3′ (SEQ ID NO:41).

The final step in the construction of the targeting vector involvedcloning the 8.8 kb SgrAI-AscI fragment containing the “long arm” andRSVLTR-HSVTK gene into the SgrAI and AscI sites of the pSP72-“shortarm”-βgal-PGKneo plasmid. This targeting vector was linearized bydigestion with either AscI or PacI before electroporation into ES cells.

Example 10 Antisense-Mediated Beer Inactivation

17-nucleotide antisense oligonucleotides are prepared in an overlappingformat, in such a way that the 5′ end of the first oligonucleotideoverlaps the translation initiating AUG of the Beer transcript, and the5′ ends of successive oligonucleotides occur in 5 nucleotide incrementsmoving in the 5′ direction (up to 50 nucleotides away), relative to theBeer AUG. Corresponding control oligonucleotides are designed andprepared using equivalent base composition but redistributed in sequenceto inhibit any significant hybridization to the coding mRNA. Reagentdelivery to the test cellular system is conducted through cationic lipiddelivery (P. L. Feigner, Proc. Natl. Acad. Sci. USA 84:7413, 1987). 2 ugof antisense oligonucleotide is added to 100 ul of reduced serum media(Opti-MEM I reduced serum media; Life Technologies, Gaithersburg Md.)and this is mixed with Lipofectin reagent (6 ul) (Life Technologies,Gaithersburg Md.) in the 100 ul of reduced serum media. These are mixed,allowed to complex for 30 minutes at room temperature and the mixture isadded to previously seeded MC3T3E21 or KS483 cells. These cells arecultured and the mRNA recovered. Beer mRNA is monitored using RT-PCR inconjunction with Beer specific primers. In addition, separateexperimental wells are collected and protein levels characterizedthrough western blot methods described in Example 4. The cells areharvested, resuspended in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl, 1mM EDTA, 1% SDS) and the soluble protein collected. This material isapplied to 10-20% gradient denaturing SDS PAGE. The separated proteinsare transferred to nitrocellulose and the western blot conducted asabove using the antibody reagents described. In parallel, the controloligonucleotides are added to identical cultures and experimentaloperations are repeated. Decrease in Beer mRNA or protein levels areconsidered significant if the treatment with the antisenseoligonucleotide results in a 50% change in either instance compared tothe control scrambled oligonucleotide. This methodology enablesselective gene inactivation and subsequent phenotype characterization ofthe mineralized nodules in the tissue culture model.

Example 11 Modeling of Sclerostin Core Region

Homology recognition techniques (e.g., PSI-BLAST (Altschul et al.,Nucleic Acids Res. 25:3389-402 (1997)), FUGUE (Shi et al., J. Mol. Biol.310:243-57 (2001)) suggested that the core region of SOST (SOST_Core)adopts a cystine-knot fold. FUGUE is a sensitive method for detectinghomology between sequences and structures. Human Chorionic Gonadotropinβ (hCG-β), for which an experimentally determined 3D structure is known,was identified by FUGUE (Shi et al., supra) as the closest homologue toof SOST_Core. Therefore, hCG-β was used as the structural template tobuild 3D models for SOST_Core.

An alignment of SOST_Core and its close homologues is shown in FIG. 7.Among the homologues shown in the alignment, only hCG-β (CGHB) had known3D structure. The sequence identity between SOST_Core and hCG-β wasapproximately 25%. Eight CYS residues were conserved throughout thefamily, emphasizing the overall structural similarity between SOST_Coreand hCG-β. Three pairs of cystines (1-5, 3-7, 4-8) formed disulfidebonds (shown with solid lines in FIG. 7) in a “knot” configuration,which was characteristic to the cystine-knot fold. An extra disulfidebond (2-6), shown as a dotted line in FIG. 7, was unique to this familyand distinguished the family of proteins from other cystine-knotfamilies (e.g., TGF-β, BMP).

SOST_Core was modeled using PDB (Berman et al., Acta Crystallogr. D.Biol. Crystallogr. 58(Pt 6 Pt1):899-907 (2002)) entry 1HCN, the 3Dstructure of hCG-β (Wu et al., Structure 2:545-58 (1994)), as thestructural template. Models were calculated with MODELER (Sali &Blundell, J. Mol. Biol. 234:779-815 (1993)). A snapshot of the bestmodel is shown in FIG. 8.

Most of the cystine-knot proteins form dimers because of the lack ofhydrophobic core in a monomer (Scheufler et al., supra; Schlunegger andGrutter, J. Mol. Biol. 231:445-58 (1993)); Wu et al., supra). SOSTlikely follows the same rule and forms a homodimer to increase itsstability. Constructing a model for the dimerized SOST_Core regionpresented several challenges because (1) the sequence similarity betweenSOST_Core and hCG-β was low (25%); (2) instead of a homodimer, hCG-βformed a heterodimer with hCG-α; and (3) a number of different relativeconformations of monomers have been observed in dimerized cystine-knotproteins from different families (e.g., PDGF, TGF-β, Neurotrophin,IL-17F, Gonadotropin), which suggested that the dimer conformation ofSOST could deviate significantly from the hCG-α/β heterodimerconformation. In constructing the model, hCG-α was replaced with hCG-βfrom the heterodimer structure (1HCN) using structure superimpositiontechniques combined with manual adjustment, and then a SOST_Corehomodimer model was built according to the pseudo hCG-β homodimerstructure. The final model is shown in FIG. 9.

Example 12 Modeling SOST-BMP Interaction

This example describes protein modeling of type I and type II receptorbinding sites on BMP that are involved with interaction between BMP andSOST.

Competition studies demonstrated that SOST competed with both type I andtype II receptors for binding to BMP. In an ELISA-based competitionassay, BMP-6 selectively interacted with the sclerostin-coated surface(300 ng/well) with high affinity (K_(D)=3.4 nM). Increasing amounts ofBMP receptor IA (FC fusion construct) competed with sclerostin forbinding to BMP-6 (11 nM) (IC₅₀=114 nM). A 10-fold molar excess of theBMP receptor was sufficient to reduce binding of sclerostin to BMP-6 byapproximately 50%. This competition was also observed with a BMPreceptor II-FC fusion protein (IC₅₀=36 nM) and DAN (IC₅₀=43 nM).Specificity of the assay was shown by lack of competition for binding toBMP-6 between sclerostin and a rActivin R1B-FC fusion protein, a TGF-βreceptor family member that did not bind BMP.

The type I and type II receptor binding sites on a BMP polypeptide havebeen mapped and were spatially separated (Scheufler et al., supra; Inniset al., supra; Nickel et al., supra; Hart et al. supra). Noggin, anotherBMP antagonist that binds to BMP with high affinity, contacts BMP atboth type I and type II receptor binding sites via the N-terminalportion of Noggin (Groppe et al., supra). The two β-strands in the coreregion near the C-terminal also contact BMP at the type II receptorbinding site.

A manually tuned alignment of Noggin and SOST indicated that the twopolypeptides shared sequence similarity between the N-terminal portionsof the proteins and between the core regions. An amino acid sequencealignment is presented in FIG. 10. The cysteine residues that form thecharacteristic cys-knot were conserved between Noggin and SOST. Theoverall sequence identity was 24%, and the sequence identity within theN-terminal binding region (alignment positions 1-45) was 33%. Tworesidues in the Noggin N-terminal binding region, namely Leu (L) atalignment position 21 and Glu (E) at position 23, were reported to playimportant roles in BMP binding (Groppe et al., supra). Both residueswere conserved in SOST as well. The sequence similarity within the coreregion (alignment positions 131-228) was about 20%, but the cys-knotscaffold was maintained and a sufficient number of key residues wasconserved, supporting homology between Noggin and SOST.

The Noggin structure was compared to SOST also to understand how twoSOST monomers dimerize. As shown in FIG. 11, the Noggin structuresuggested that the linker between the N-terminal region and the coreregion not only played a role in connecting the two regions, but alsoformed part of the dimerization interface between two Noggin monomers.One major difference between Noggin and SOST was that the linker betweenthe N-terminal region and the core region was much shorter in SOST.

The C-terminal region of SOST may play a role in SOST dimerization. Thesequence of Noggin ended with the core region, while SOST had an extraC-terminal region. In the Noggin structure a disulfide bond connectedthe C-termini of two Noggin monomers. Thus, the C-terminal region ofSOST started close to the interface of two monomers and could contributeto dimerization. In addition, secondary structure prediction showed thatsome portions of the C-terminal region of SOST had a tendency to formhelices. This region in SOST may be responsible for the dimerizationactivity, possibly through helix-helix packing, which mimicked thefunction of the longer linker in Noggin. Another difference between thestructure of Noggin and SOST was the amino acid insertion in the SOSTcore region at alignment positions 169-185 (see FIG. 10). This insertionextended a β-hairpin, which pointed towards the dimerization interfacein the Noggin structure (shown in FIG. 11 as a loop region in the middleof the monomers and above the C-terminal Cys residue). This elongatedβ-hairpin could also contribute to SOST dimerization.

Example 13 Design and Preparation of SOST Peptide Immunogens

This Example describes the design of SOST peptide immunogens that areused for immunizing animals and generating antibodies that blockinteractions between BMP and SOST and prevent dimer formation of SOSTmonomers.

BMP Binding Fragments

The overall similarity between SOST and Noggin and the similaritybetween the N-terminal regions of the two polypeptides suggest that SOSTmay interact with BMP in a similar manner to Noggin. That is, theN-terminal region of SOST may interact with both the type I and type IIreceptor binding sites on BMP, and a portion of the core region (aminoacid alignment positions 190-220 in FIG. 10) may interact with the typeII receptor binding site such that antibodies specific for these SOSTregions may block or impair binding of BMP to SOST.

The amino acid sequences of these SOST polypeptide fragments for rat andhuman SOST are provided as follows.

-   -   SOST_N_Linker: The N-terminal region (includes the short linker        that connects to the core region)

Human: (SEQ ID NO: 92)QGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETK DVSEYS Rat: (SEQ IDNO: 93) QGWQAFKNDATEIIPGLREYPEPPQELENNQTMNRAENGGRPPHHPYDTK DVSEYS

-   -   SOST_Core_Bind: Portion of the core region that is likely to        contact BMP at its type II receptor binding site (extended        slightly at both termini to include the CYS residue anchors):

(SEQ ID NO: 94) Human: CIPDRYRAQRVQLLCPGGEAPRARKVRLVASC (SEQ ID NO: 95)Rat: CIPDRYRAQRVQLLCPGGAAPRSRKVRLVASC

SOST Dimerization Fragments

The C-terminal region of SOST is likely to be involved in the formationof SOST homodimers (see Example 12). The elongated β-hairpin may alsoplay a role in homodimer formation. Antibodies that specifically bind tosuch regions may prevent or impair dimerization of SOST monomers, whichmay in turn interfere with interaction between SOST and BMP. Polypeptidefragments in rat and human SOST corresponding to these regions are asfollows.

SOST C: the C-Terminal Region

Human: (SEQ ID NO: 96) LTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY Rat:(SEQ ID NO: 97) LTRFHNQSELKDFGPETARPQKGRPRPRARGAKANQAELENAY

SOST_Core_Dimer: Portion of the core region that is likely involved inSOST dimerization (extended slightly at both termini to include the Cysresidue anchors):

Human: CGPARLLPNAIGRGKWWRPSGPDFRC (SEQ ID NO: 98) Rat:CGPARLLPNAIGRVKWWRPNGPDFRC (SEQ ID NO: 99)

BMP Binding Fragment at SOST N-Terminus

The key N-terminal binding region of SOST (alignment positions 1-35 inFIG. 10) was modeled on the basis of the Noggin/BMP-7 complex structure(Protein Data Bank Entry No: 1M4U) and the amino acid sequence alignment(see FIG. 10) to identify amino acid residues of the SOST N-terminusthat likely interact with BMP. The model of SOST is presented in FIG.12. In the comparative model, phenylalanine (Phe, F) at alignmentposition 8 (see arrow and accompanying text) in the SOST sequenceprojects into a hydrophobic pocket on the surface of the BMP dimer. Thesame “knob-into-hole” feature has been observed in the BMP and type Ireceptor complex structure (Nickel et al., supra), where Phe85 of thereceptor fits into the same pocket, which is a key feature inligand-type I receptor recognition for TGF-β superfamily members(including, for example, TGF-β family, BMP family, and the like).According to the model, a proline (Pro) directed turn is also conserved,which allows the N-terminal binding fragment to thread along the BMPdimer surface, traveling from type I receptor binding site to type IIreceptor binding site on the other side of the complex. Also conservedis another Pro-directed turn near the carboxy end of the bindingfragment, which then connects to the linker region. Extensive contactsbetween SOST and BMP are evident in FIG. 12.

Peptide Immunogens

Peptides were designed to encompass the SOST N-terminal region predictedto make contact with BMP proteins. The peptide sequences are presentedbelow. For immunizing animals, the peptide sequences were designed tooverlap, and an additional cysteine was added to the C-terminal end tofacilitate crosslinking to KLH. The peptides were then used forimmunization. The peptide sequences of the immunogens are as follows.

Human SOST:

QGWQAFKNDATEIIPELGEY (SEQ ID NO: 47) TEIIPELGEYPEPPPELENN (SEQ ID NO:48) PEPPPELENNKTMNRAENGG (SEQ ID NO: 49) KTMNRAENGGRPPHHPFETK (SEQ IDNO: 50) RPPHHPFETKDVSEYS (SEQ ID NO: 51)

Human SOST Peptides with Additional Cys:

QGWQAFKNDATEIIPELGEY-C (SEQ ID NO: 52) TEIIPELGEYPEPPPELENN-C (SEQ IDNO: 53) PEPPPELENNKTMNRAENGG-C (SEQ ID NO: 54) KTMNRAENGGRPPHHPFETK-C(SEQ ID NO: 55) RPPHHPFETKDVSEYS-C (SEQ ID NO: 56)

Rat SOST:

QGWQAFKNDATEIIPGLREYPEPP (SEQ ID NO: 57) PEPPQELENNQTMNRAENGG (SEQ IDNO: 58) ENGGRPPHHPYDTKDVSEYS (SEQ ID NO: 59) TEIIPGLREYPEPPQELENN (SEQID NO: 60)

Rat SOST Peptides with Additional Cys:

QGWQAFKNDATEIIPGLREYPEPP-C (SEQ ID NO: 61) PEPPQELENNQTMNRAENGG-C (SEQID NO: 62) ENGGRPPHHPYDTKDVSEYS-C (SEQ ID NO: 63) TEIIPGLREYPEPPQELENN-C(SEQ ID NO: 64)

The following peptides were designed to contain the amino acid portionof core region that was predicted to make contact with BMP proteins.Cysteine was added at the C-terminal end of each peptide for conjugationto KLH, and the conjugated peptides were used for immunization. In theDocking Core N-terminal Peptide an internal cysteine was changed to aserine to avoid double conjugation to KLH.

For Human SOST:

Amino Acid Sequence Without Cys Residues Added:

Docking_Core_N-terminal_Peptide: IPDRYRAQRVQLLCPGGEAP (SEQ ID NO: 66)Docking_Core_Cterm_Peptide: QLLCPGGEAPRARKVRLVAS (SEQ ID NO: 67)Docking_Core_N-terminal_Peptide: IPDRYRAQRVQLLCPGGEAP-C (SEQ ID NO: 68)Docking_Core_Cterm_Peptide: QLLCPGGEAPRARKVRLVAS-C (SEQ ID NO: 69)

For Rat SOST:

Amino Acid Sequence Without Cys Residues Added or Substituted:

Docking_Core_N-terminal_Peptide: IPDRYRAQRVQLLSPGG (SEQ ID NO: 70)Docking_Core_Cterm_Peptide: PGGAAPRSRKVRLVAS (SEQ ID NO: 71)

Peptide Immunogens with Cys Added and Substituted:

Docking_Core_N-terminal_Peptide: IPDRYRAQRVQLLSPGG-C (SEQ ID NO: 72)Docking_Core_Cterm_Peptide: PGGAAPRSRKVRLVAS-C (SEQ ID NO: 73)

Two regions within SOST that potentially interact to form SOSThomodimers include the amino acids with the SOST core region that arenot present in Noggin. Human SOST peptides designed to contain thissequence had a C-terminal or N-terminal Cys that was conjugated to KLH.For the rat SOST peptide, a cysteine was added to the carboxy terminusof the sequence (SEQ ID NO:76). The KLH conjugated peptides were usedfor immunization.

For Human SOST:

CGPARLLPNAIGRGKWWRPS (SEQ ID NO: 74) IGRGKWWRPSGPDFRC (SEQ ID NO: 75)

For Rat SOST:

PNAIGRVKWWRPNGPDFR (SEQ ID NO:76)

Rat SOST Peptide with Cysteine Added

PNAIGRVKWWRPNGPDFR-C (SEQ ID NO: 77)

The second region within SOST that potentially interacts to form SOSThomodimers includes the C-terminal region. Peptide immunogens weredesigned to include amino acid sequences within this region (see below).For conjugation to KLH, a cysteine residue was added to the C-terminalend, and the conjugated peptides were used for immunization.

For Human SOST:

KRLTRFHNQS ELKDFGTEAA (SEQ ID NO: 78) ELKDFGTEAA RPQKGRKPRP (SEQ ID NO:79) RPQKGRKPRP RARSAKANQA (SEQ ID NO: 80) RARSAKANQA ELENAY (SEQ ID NO:81)

Peptide Immunogens with Cys Added at C-Terminus:

KRLTRFHNQS ELKDFGTEAA-C (SEQ ID NO: 82) ELKDFGTEAA RPQKGRKPRP-C (SEQ IDNO: 83) RPQKGRKPRP RARSAKANQA-C (SEQ ID NO: 84) RARSAKANQA ELENAY-C (SEQID NO: 85)

For Rat SOST:

KRLTRFHNQSELKDFGPETARPQ (SEQ ID NO: 86) KGRKPRPRARGAKANQAELENAY (SEQ IDNO: 87) SELKDFGPETARPQKGRKPRPRAR (SEQ ID NO: 88)

Peptide Immunogens with Cys Added at C-Terminus:

KRLTRFHNQSELKDFGPETARPQ-C (SEQ ID NO: 89) KGRKPRPRARGAKANQAELENAY-C (SEQID NO: 90) SELKDFGPETARPQKGRKPRPRAR-C (SEQ ID NO: 91)

Example 14 Assay for Detecting Binding of Antibodies to a TGF-BetaBinding-Protein

This example describes an assay for detecting binding of a ligand, forexample, an antibody or antibody fragment thereof, to sclerostin.

A FLAG®-sclerostin fusion protein was prepared according to protocolsprovided by the manufacturer (Sigma Aldrich, St. Louis, Mo.) and asdescribed in U.S. Pat. No. 6,395,511. Each well of a 96 well microtiterplate is coated with anti-FLAG® monoclonal antibody (Sigma Aldrich) andthen blocked with 10% BSA in PBS. The fusion protein (20 ng) is added to100 μl PBS/0.2% BSA and adsorbed onto the 96-well plate for 60 minutesat room temperature. This protein solution is removed and the wells arewashed to remove unbound fusion protein. A BMP, for example, BMP-4,BMP-5, BMP-6, or BMP-7, is diluted in PBS/0.2% BSA and added to eachwell at concentrations ranging from 10 μM to 500 nM. After an incubationfor 2 hours at room temperature; the binding solution is removed and theplate is washed three times with 200 μl volumes of PBS/0.2% BSA. Bindingof the BMP to sclerostin is detected using polyclonal antiserum ormonoclonal antibody specific for the BMP and an appropriateenzyme-conjugated second step reagent according to standard ELISAtechniques (see, e.g., Ausubel et al., Current Protocols in Mol Biol.Vol 2 11.2.1-11.2.22 (1998)). Specific binding is calculated bysubtracting non-specific binding from total binding and analyzed usingthe LIGAND program (Munson and Podbard, Anal. Biochem. 107:220-39(1980)).

Binding of sclerostin to a BMP is also detected by homogeneous timeresolved fluorescence detection (Mellor et al., J Biomol. Screening,3:91-99 (1998)). A polynucleotide sequence encoding sclerostin isoperatively linked to a human immunoglobulin constant region in arecombinant nucleic acid construct and expressed as a humanFc-sclerostin fusion protein according to methods known in the art anddescribed herein. Similarly, a BMP ligand is engineered and expressed asa BMP-mouse Fc fusion protein. These two fusion proteins are incubatedtogether and the assay conducted as described by Mellor et al.

Example 15 Screening Assay for Antibodies that Inhibit Binding ofTGF-Beta Family Members to TGF-Beta Binding Protein

This example describes a method for detecting an antibody that inhibitsbinding of a TGF-beta family member to sclerostin. An ELISA is performedessentially as described in Example 14 except that the BMP concentrationis held fixed at its Kd (determined, for example, by BIAcore analysis).In addition, an antibody or a library or to collection of antibodies isadded to the wells to a concentration of 1 μM. Antibodies are incubatedfor 2 hours at room temperature with the BMP and sclerostin, thesolution removed, and the bound BMP is quantified as described (seeExample 14). Antibodies that inhibit 40% of the BMP binding observed inthe absence of antibody are considered antagonists of this interaction.These antibodies are further evaluated as potential inhibitors byperforming titration studies to determine their inhibition constants andtheir effect on TGF-beta binding-protein binding affinity. Comparablespecificity control assays may also be conducted to establish theselectivity profile for the identified antagonist using assays dependenton the BMP ligand action (e.g., a BMP/BMP receptor competition study).

Example 16 Inhibition of TGF-Beta Binding-Protein Localization to BoneMatrix

Evaluation of inhibition of localization to bone matrix (hydroxyapatite)is conducted using modifications to the method of Nicolas (Calcif.Tissue Int. 57:206-12 (1995)). Briefly, ¹²⁵I-labelled TGF-betabinding-protein is prepared as described by Nicolas (supra).Hydroxyapatite is added to each well of a 96-well microtiter plateequipped with a polypropylene filtration membrane (Polyfiltroninc,Weymouth Mass.). TGF-beta binding-protein diluted in 0.2% albumin in PBSbuffer is then added to the wells. The wells containing matrix arewashed 3 times with 0.2% albumin in PBS buffer. Adsorbed TGF-betabinding-protein is eluted using 0.3 M NaOH and then quantified.

An antibody that inhibits or impairs binding of the sclerostin TGF-betabinding protein to the hydroxyapatite is identified by incubating theTGF-beta binding protein with the antibody and applying the mixture tothe matrix as described above. The matrix is washed 3 times with 0.2%albumin in PBS buffer. Adsorbed sclerostin is eluted with 0.3 M NaOH andthen quantified. An antibody that inhibits the level of binding ofsclerostin to the hydroxyapatite by at least 40% compared to the levelof binding observed in the absence of antibody is considered a bonelocalization inhibitor. Such an antibody is further characterized indose response studies to determine its inhibition constant and itseffect on TGF-beta binding-protein binding affinity.

From the foregoing, although specific embodiments of the invention havebeen described herein for purposes of illustration, variousmodifications may be made without deviating from the spirit and scope ofthe invention. Accordingly, the invention is not limited except as bythe appended claims.

1-20. (canceled)
 21. An isolated polynucleotide encoding an antibody, orantigen binding fragment thereof, that specifically binds to an epitopeof amino acids 86-111 of SEQ ID NO:
 46. 22. The polynucleotide encodingthe antibody of claim 21, wherein the antibody, or an antigen-bindingfragment thereof, specifically binds to an epitope of amino acids 86-105of SEQ ID NO:
 46. 23. The polynucleotide encoding the antibody of claim21, wherein the antibody, or an antigen-binding fragment thereof,specifically binds to an epitope of amino acids 96-111 of SEQ ID NO: 46.24. The polynucleotide encoding the antibody of claim 21, wherein theantibody, or an antigen-binding fragment thereof, specifically binds toan epitope of amino acids 93-110 of SEQ ID NO:
 46. 25. Thepolynucleotide encoding the antibody of claim 21, wherein the antibody,or antigen binding fragment thereof, binds to a peptide consisting ofamino acids 86-111 of SEQ ID NO: 46 with an affinity K_(D) of less thanor equal to about 10⁻⁷ M.
 26. The polynucleotide encoding the antibodyof claim 21, wherein the antibody is a human antibody, a humanizedantibody or a chimeric antibody.
 27. The polynucleotide encoding theantibody of claim 25, wherein the antibody is a human antibody, ahumanized antibody or a chimeric antibody.
 28. The polynucleotideencoding the antibody of claim 21, wherein the antibody is anantigen-binding fragment.
 29. The polynucleotide encoding the antibodyof claim 25, wherein the antibody is an antigen-binding fragment. 30.The polynucleotide encoding the antibody of claim 21, wherein theantibody comprises an antigen-binding fragment is selected from thegroup consisting of F(ab′)₂, Fab, Fab, Fd, and Fv.
 31. Thepolynucleotide encoding the antibody of claim 21, wherein the antibodycomprises an Fv fragment.
 32. The polynucleotide encoding the antibodyof claim 21 wherein the antibody comprises a single chain antibody. 33.The polynucleotide encoding the antibody of claim 21, wherein theantibody comprises at least one heavy chain and at least one lightchain.
 34. The polynucleotide encoding the antibody of claim 25, whereinthe antibody comprises at least one heavy chain and at least one lightchain.
 35. An isolated polynucleotide encoding a heavy chain of anantibody, or antigen binding fragment thereof, that specifically bindsto an epitope of amino acids 86-111 of SEQ ID NO:
 46. 36. An isolatedpolynucleotide encoding a light chain of an antibody, or antigen bindingfragment thereof, that specifically binds to an epitope of amino acids86-111 of SEQ ID NO:
 46. 37. A vector comprising the polynucleotide ofany one of claims 21-36, operably linked to a transcriptional ortranslational regulatory element.
 38. A host cell comprising an isolatedpolynucleotide of any of claims 21-36.
 39. A host cell comprising theisolated polynucleotide of claim 35 and the isolated polynucleotide ofclaim
 36. 40. A host cell comprising a vector of claim
 37. 41. A hostcell that is capable of expressing an antibody, or antigen bindingfragment thereof, that specifically binds to an epitope of amino acids86-111 of SEQ ID NO:
 46. 42. A method of producing an antibodycomprising the step of culturing the host cell of claim 38 and isolatingthe antibody produced therefrom.
 43. A method of producing an antibodycomprising the step of culturing the host cell of claim 39 and isolatingthe antibody produced therefrom.
 44. A method of producing an antibodycomprising the step of culturing the host cell of claim 40 and isolatingthe antibody produced therefrom.
 45. A method of producing an antibodycomprising the step of culturing the host cell of claim 41 and isolatingthe antibody produced therefrom.